Genomic features of an extensively drug-resistant and NDM-1-positive Klebsiella pneumoniae ST340 isolated from river water.

The environmental contamination plays a significant role in the emergence of antimicrobial resistance. In this study, we report a genomic analysis of an extensively drug-resistant and blaNDM-1-producing Klebsiella pneumoniae (EW807) strain recovered from a surface water sample. Strain EW807 belonged to sequence type (ST) 340 and serotype O4:KL15, a high-risk clone of the clonal group 258. This strain carried a broad resistome, including blaNDM-1 and blaCTX-M-15. The core genome multilocus sequence typing phylogenetic analysis revealed that the EW807 strain was most related to strains from Brazil and the USA. An IncX3 plasmid was identified harboring the blaNDM-1 gene, while an IncFIB(K) plasmid was detected carrying the blaCTX-M-15 in addition to multidrug resistance and multimetal tolerance regions. IncX3 and IncFIB(K) plasmids shared high similarity with plasmids from a human in China and a dog in Brazil, respectively. The regions harboring the blaNDM-1 and blaCTX-M-15 genes contained sequences from the Tn3 family. These findings suggest that IncX3 plasmid could play a role in the spread of NDM-1 in a post-pandemic scenario. To the best of our knowledge, this is the first report of blaNDM-1-producing K. pneumoniae ST340 O4:KL15 strain in the environment. Therefore, the presence of high-risk clones of K. pneumoniae carrying carbapenemases in the environment requires strict surveillance.

Klebsiella pneumoniae bacteraemia epidemiology: resistance profiles and clinical outcome of King Fahad Medical City isolates, Riyadh, Saudi Arabia.

BACKGROUND AND OBJECTIVES: Klebsiella pneumoniae (K. pneumoniae) is the second leading cause of community-acquired and hospital-acquired gram-negative bloodstream infection (BSI). This study aimed to assess the epidemiological and microbial-resistance characteristics and clinical factors associated with K. pneumoniae BSI in Saudi Arabia. MATERIALS AND METHODS: Data of 152 K. pneumoniae isolates diagnosed between January 2019 and January 2020 at King Fahad Medical City, Riyadh, Saudi Arabia were evaluated retrospectively. Clinical records of the patients were collected and analysed statistically. RESULTS: In total, 152 cases of K. pneumoniae BSI were identified. Adult patients (66.4%) were at a higher risk of developing the infection than paediatric patients (33.6%). The rate of infection was slightly higher in women than in men. Neurological disorders were the predominant underlying conditions for the acquisition of K. pneumoniae BSI, at all ages. Most of the deceased patients were adults with multi-organ dysfunction. Klebsiella pneumoniae showed disturbing resistance to amoxicillin-clavulanate and cefuroxime (72.4%), ceftazidime (67.8), cephalothin (76.3%), and to Carbapenems (36.1%). CONCLUSIONS: The impact of K. pneumoniae BSI was seen not only at the patient level, but also at the community level, and was related to multi-drug resistant infection. These findings provide a better understanding of microbial resistance and its association with patient clinical outcomes.

A SERS bioassay based on vancomycin-modified PEI-interlayered nanocomposite and aptamer-functionalized SERS tags for synchronous detection of Acinetobacter baumannii and Klebsiella pneumoniae.

Klebsiella pneumoniae (KP) and Acinetobacter baumannii (AB) are two important gram-negative bacteria that cause pneumonia and have been recently known to be associated with food. The rapid detection of these pathogens in food is important to minimize their colonization of the gut and stop new threats of the disease from spreading across the food chain. Herein, a double-edged sword aptasensor was developed for the synchronous detection of KP and AB in food and clinical samples. A highly sensitive, selective, specific, and synchronous detection of the target bacteria was achieved, and the limit of detection (LOD) was 10 cells/mL with a liner range of 50 to 105 cells/mL. The total assay time was 1.5 h. This study does not only provide a new tool for the detection of the target bacteria, but also serves as a promising tool for food safety and pneumonia diagnosis.

Clinical and microbiological characteristics of bloodstream infection caused by Klebsiella pneumoniae harboring rmpA in Japanese adults.

We investigated the clinical features of bloodstream infections (BSIs) caused by Klebsiella pneumoniae harboring rmpA and molecular characteristics of the bacteria. We retrospectively investigated adult patients with K. pneumoniae BSI from January 2010 to March 2021 at Nagasaki University Hospital. A matched case-control study in a 1:3 ratio was conducted to clarify the clinical and bacterial characteristics of BSI caused by rmpA-positive K. pneumoniae compared with those caused by rmpA-negative isolates. Antimicrobial susceptibility testing and multilocus sequence typing (MLST) were performed for rmpA-positive isolates. The rmpA was detected in 36 (13.4%) of the 268 isolates. Of these 36 isolates, 31 (86.1%) harbored iucA and 35 (97.2%) each possessed peg-344 and iroB; capsular types were identified as K1 in 9 (25.0%) and K2 in 10 isolates (27.8%). Contrarily, of the 108 rmpA-negative isolates, which were matched for case-control studies, 5 isolates (4.6%) harbored iucA and 1 (0.9%) each possessed peg-344 and iroB; 2 (1.9%) and 3 isolates (2.8%) had K1 and K2 capsular types, respectively. Among the rmpA-positive isolates, ST23/K1 (eight isolates) was the most frequent, followed by ST412/non-K1/K2 (seven isolates), ST86/K2 (five isolates), and ST268/non-K1/K2 (four isolates). In a multivariate analysis using clinical factors, liver abscess positively correlated with rmpA-positive isolates, whereas biliary tract infection and use of anticancer drugs negatively correlated with rmpA-positive isolates in patients with K. pneumoniae BSI. Considering the correlation between rmpA-positive isolates and clinical features, rmpA can be used as a marker for understanding the pathophysiology of K. pneumoniae BSI.

Detection of carbapenemase- and ESBL-producing Klebsiella pneumoniae from bovine bulk milk and comparison with clinical human isolates in Italy.

Klebsiella pneumoniae is the most common Klebsiella species infecting animals and is one of the causing agents of mastitis in cows. The rise of antimicrobial resistance in K. pneumoniae, particularly in strains producing extended-spectrum ß-lactamases (ESBLs) and/or carbapenemases, is of concern worldwide. Recently (Regulation UE No 2022/1255), carbapenems and cephalosporins in combination with ß-lactamase inhibitors have been reserved only to human treatments in the European Union. The aim of this study was to investigate the role of cattle as carrier of human pathogenic carbapenem-resistant (CR) and ESBL-producing K. pneumoniae. On this purpose, a study involving 150 dairy farms in Parma province (Northern Italy) and 14 non replicate K. pneumoniae isolates from patients admitted at Parma University-Hospital was planned. Four multidrug resistant (MDR) K. pneumoniae strains were detected from 258 milk filters collected between 2019 and 2021. One carbapenemase KPC-3-positive K. pneumoniae ST307 (0.4 %; 95 % CI - 0.07 - 2.2) was detected in milk filters. The isolate also harboured OXA-9, CTX-M-15 and SHV-106 determinants, together with genes conferring resistance to aminoglycosides (aac(3')-IIa, aph (3″)-Ib, aph (6)-Id), fluoroquinolones (oqxA, oqxB, qnrB1), phosphonic acids (fosA6), sulphonamides (sul2), tetracyclines (tet(A)6) and trimethoprim (dfrA14). One KPC-3-producing K. pneumoniae ST307 was identified also among the human isolates, thus suggesting a possible circulation of pathogens out of the clinical settings. The remaining three bovine isolates were MDR ESBL-producing K. pneumoniae characterized by different genomic profiles: CTX-M-15, TEM-1B and SHV-187 genes (ST513); CTX-M-15 and SHV-145 (ST307); SHV-187 and DHA-1 (ST307). Occurrence of ESBL-producing K. pneumoniae in milk filters was 1.2 % (95 % CI 0.4-3.4). All the isolates showed resistance to aminoglycosides, 3rd-generation cephalosporins, and fluoroquinolones. Among the human isolates, two multidrug resistant ESBL-producing K. pneumoniae ST307 were found, thus confirming the circulation of this high-risk lineage between humans and cattle. Our findings suggest that food-producing animals can carry human pathogenic microorganisms harboring resistance genes against carbapenems and 3rd-generation cephalosporins, even if not treated with such antimicrobials. Moreover, on the MDR K. pneumoniae farms, the antimicrobial use was much higher than the Italian median value, thus highlighting the importance of a more prudent use of antibiotics in animal productions.

Pyogenic Liver Abscess: Contemporary Trends in a Tertiary Institute.

Background: Pyogenic liver abscess (PLA) is an uncommon but potentially life-threatening condition. In recent years, advances in diagnostics and management have led to early diagnosis and treatment and decreased mortality. We present recent data from a large series of patients with PLA and examine the trends in the management of PLA over a period of 50 years. Methods: The medical records of all patients admitted to the Shaare Zedek Medical Center, Israel, between January 2011 and December 2021 with a primary or secondary diagnosis of PLA were reviewed retrospectively. Results: : Ninety-five patients with PLA were identified. Thirty-eight (40%) were female. The median patient age was 66 years (range 18-93). The diagnosis of PLA in all patients was confirmed with abdominal computed tomography (CT). In twenty patients (21.1%), PLA was not diagnosed by the initial abdominal US. Most abscesses were right-sided. Biliary tract origin was the most common underlying cause of PLA (n = 57, 60%), followed by cryptogenic etiology (n = 28, 30%). Escherichia coli, Klebsiella pneumoniae, and Streptococcus species were most commonly identified. The most common primary treatment modality was percutaneous drainage (PD), which was performed in 81 patients (85.3%). Fourteen patients (14.7%) were treated medically without intervention, and two patients (2.1%) were treated surgically following a failure of PD. Four patients died as a direct result of PLA. Conclusions: Patients diagnosed with PLA are older, the male predominance is less pronounced, and the offending pathogens are likely to originate from the biliary tract. This study questions the utility of abdominal US as the initial diagnostic imaging in patients with suspected PLA (versus CT) and demonstrates improved outcomes for patients with PLA over the years.

Polymyxin Resistance in Clinical Isolates of K. pneumoniae in Brazil: Update on Molecular Mechanisms, Clonal Dissemination and Relationship With KPC-Producing Strains.

In Brazil, the production of KPC-type carbapenemases in Enterobacteriales is endemic, leading to widespread use of polymyxins. In the present study, 502 Klebsiella pneumoniae isolates were evaluated for resistance to polymyxins, their genetic determinants and clonality, in addition to the presence of carbapenem resistance genes and evaluation of antimicrobial resistance. Resistance to colistin (polymyxin E) was evaluated through initial selection on EMB agar containing 4% colistin sulfate, followed by Minimal Inhibitory Concentration (MIC) determination by broth microdilution. The susceptibility to 17 antimicrobials was assessed by disk diffusion. The presence of blaKPC, blaNDM and blaOXA-48-like carbapenemases was investigated by phenotypic methods and conventional PCR. Molecular typing was performed by PFGE and MLST. Allelic variants of the mcr gene were screened by PCR and chromosomal mutations in the pmrA, pmrB, phoP, phoQ and mgrB genes were investigated by sequencing. Our work showed a colistin resistance frequency of 29.5% (n = 148/502) in K. pneumoniae isolates. Colistin MICs from 4 to >128 µg/mL were identified (MIC50 = 64 µg/mL; MIC90 >128 µg/mL). All isolates were considered MDR, with the lowest resistance rates observed for amikacin (34.4%), and 19.6% of the isolates were resistant to all tested antimicrobials. The blaKPC gene was identified in 77% of the isolates, in consonance with the high rate of resistance to polymyxins related to its use as a therapeutic alternative. Through XbaI-PFGE, 51 pulsotypes were identified. MLST showed 21 STs, with ST437, ST258 and ST11 (CC11) being the most prevalent, and two new STs were determined: ST4868 and ST4869. The mcr-1 gene was identified in 3 K. pneumoniae isolates. Missense mutations in chromosomal genes were identified, as well as insertion sequences in mgrB. Furthermore, the identification of chromosomal mutations in K. pneumoniae isolates belonging from CC11 ensures its success as a high-risk epidemic clone in Brazil and worldwide.

Genomic surveillance for multidrug-resistant or hypervirulent Klebsiella pneumoniae among United States bloodstream isolates.

BACKGROUND: Klebsiella pneumoniae strains have been divided into two major categories: classical K. pneumoniae, which are frequently multidrug-resistant and cause hospital-acquired infections in patients with impaired defenses, and hypervirulent K. pneumoniae, which cause severe community-acquired and disseminated infections in normal hosts. Both types of infections may lead to bacteremia and are associated with significant morbidity and mortality. The relative burden of these two types of K. pneumoniae among bloodstream isolates within the United States is not well understood. METHODS: We evaluated consecutive K. pneumoniae isolates cultured from the blood of hospitalized patients at Northwestern Memorial Hospital (NMH) in Chicago, Illinois between April 2015 and April 2017. Bloodstream isolates underwent whole genome sequencing, and sequence types (STs), capsule loci (KLs), virulence genes, and antimicrobial resistance genes were identified in the genomes using the bioinformatic tools Kleborate and Kaptive. Patient demographic, comorbidity, and infection information, as well as the phenotypic antimicrobial resistance of the isolates were extracted from the electronic health record. Candidate hypervirulent isolates were tested in a murine model of pneumonia, and their plasmids were characterized using long-read sequencing. We also extracted STs, KLs, and virulence and antimicrobial resistance genes from the genomes of bloodstream isolates submitted from 33 United States institutions between 2007 and 2021 to the National Center for Biotechnology Information (NCBI) database. RESULTS: Consecutive K. pneumoniae bloodstream isolates (n = 104, one per patient) from NMH consisted of 75 distinct STs and 51 unique capsule loci. The majority of these isolates (n = 58, 55.8%) were susceptible to all tested antibiotics except ampicillin, but 17 (16.3%) were multidrug-resistant. A total of 32 (30.8%) of these isolates were STs of known high-risk clones, including ST258 and ST45. In particular, 18 (17.3%) were resistant to ceftriaxone (of which 17 harbored extended-spectrum beta-lactamase genes) and 9 (8.7%) were resistant to meropenem (all of which harbored a carbapenemase genes). Four (3.8%) of the 104 isolates were hypervirulent K. pneumoniae, as evidenced by hypermucoviscous phenotypes, high levels of virulence in a murine model of pneumonia, and the presence of large plasmids similar to characterized hypervirulence plasmids. These isolates were cultured from patients who had not recently traveled to Asia. Two of these hypervirulent isolates belonged to the well characterized ST23 lineage and one to the re-emerging ST66 lineage. Of particular concern, two of these isolates contained plasmids with tra conjugation loci suggesting the potential for transmission. We also analyzed 963 publicly available genomes of K. pneumoniae bloodstream isolates from locations within the United States. Of these, 465 (48.3%) and 760 (78.9%) contained extended-spectrum beta-lactamase genes or carbapenemase genes, respectively, suggesting a bias towards submission of antibiotic-resistant isolates. The known multidrug-resistant high-risk clones ST258 and ST307 were the predominant sequence types. A total of 32 (3.3%) of these isolates contained aerobactin biosynthesis genes and 26 (2.7%) contained at least two genetic features of hvKP strains, suggesting elevated levels of virulence. We identified 6 (0.6%) isolates that were STs associated with hvKP: ST23 (n = 4), ST380 (n = 1), and ST65 (n = 1). CONCLUSIONS: Examination of consecutive isolates from a single center demonstrated that multidrug-resistant high-risk clones are indeed common, but a small number of hypervirulent K. pneumoniae isolates were also observed in patients with no recent travel history to Asia, suggesting that these isolates are undergoing community spread in the United States. A larger collection of publicly available bloodstream isolate genomes also suggested that hypervirulent K. pneumoniae strains are present but rare in the USA; however, this collection appears to be heavily biased towards highly antibiotic-resistant isolates (and correspondingly away from hypervirulent isolates).

Molecular characterization of extended-spectrum ß-lactamase producers, carbapenemase producers, polymyxin-resistant, and fosfomycin-resistant Enterobacterales among pigs from Egypt.

OBJECTIVES: To perform the first prospective surveillance evaluating the occurrence of genes encoding colistin resistance, fosfomycin resistance, carbapenemase, or extended-spectrum ß-lactamases (ESBLs) among Enterobacterial isolates recovered from the gut flora of pigs from Egypt. METHODS: Between February and April 2020, 81 rectal swabs were collected from pigs in a slaughterhouse, Cairo, Egypt. Samples were screened for different resistance mechanisms using SuperPolymyxin, ChromID ESBL, SuperFOS, and SuperCarba selective agar plates. Antimicrobial susceptibility testing was performed for all isolates using disk diffusion and broth microdilution techniques. PCR screening was performed for ESBLs, carbapenemases, mcr, and fosA genes. Mating-out assays, multilocus sequence typing analysis, and plasmid typing were also performed. RESULTS: A high prevalence of ESBLs, carbapenemases, fosfomycin, and colistin resistance genes was evidenced among those isolates. The predominant ESBL identified was blaCTX-M-15, followed by blaCTX-M-9. We also identified blaNDM-5 and blaOXA-244. fosA3, fosA4, and fosA6 were identified in E. coli isolates. In addition, 11 MCR-1 producers were recovered. Notably, co-occurrence of ESBL genes and mcr or fosA genes was observed. MLST analysis revealed a high clonal diversity, ruling out the dissemination of one major clone. IncFIB-type was predominantly present among ESBL and FosA producers. The blaNDM-5 gene was carried on an IncX4-type, although the blaOXA-244 gene was chromosomally located. The mcr-1 gene was carried on a diversity of plasmids (IncI2, IncX4, and IncHI2). CONCLUSION: These results raise serious public health concerns as Egyptian pig meat could serve as a reservoir for antimicrobial resistance genes (ARGs), leading to worldwide dissemination.

Genomic insights into virulence factors affecting tissue-invasive Klebsiella pneumoniae infection.

BACKGROUND: The key virulence factors responsible for hypervirulent Klebsiella pneumoniae (hvKp) infection remains elusive. METHODS: We analyzed K. pneumoniae isolates collected between 2017 and 2019 and defined hvKp as a pyogenic infection. Classical K. pneumoniae (cKp) involved a non-invasive infection or uncomplicated bacteremia. Isolates belonging to the K. pneumoniae species complex were excluded. RESULTS: We analyzed 112 isolates, including 19 hvKp, 67 cKp, and 26 colonizers, using whole-genome sequencing. Population genomics revealed that the K1-sequence type (ST) 82 (O1v1) clade was distinct from that of the K1-ST23 (O1v2) clone. The virulence gene profiles also differed between K1-ST82 (aerobactin and rmpA) and K1-ST23 (aerobactin, yersiniabactin, salmochelin, colibactin, and rmpA/rmpA2). The K2 genotype was more diverse than that of K1. A neighboring subclade of K1-ST23 (comprising ST29, ST412, ST36, and ST268) showed multidrug resistance and hypervirulence potentials. Logistic-regression analysis revealed that diabetes mellitus was associated with K. pneumoniae infection (odds ratio [OR]: 4.11; 95% confidence interval [CI]: 1.14-14.8). No significant association was found between hvKp diagnosis and clinical characteristics, such as diabetes mellitus or community acquisition. However, the K1 genotype (OR: 9.02; 95% CI: 2.49-32.7; positive-likelihood ratio [LR]: 4.08), rmpA (OR: 8.26; 95% CI: 1.77-38.5; positive LR: 5.83), and aerobactin (OR: 4.59; 95% CI: 1.22-17.2; positive LR: 3.49) were substantial diagnostic predictors of hvKp. CONCLUSIONS: The K1 genotype, rmpA, and aerobactin are prominent predictors of hvKp, suggesting that further pyogenic (metastatic) infection should be examined clinically. These findings may shed light on key hvKp virulence factors.

Emergence of polymyxin B-heteroresistant hypervirulent Klebsiella pneumoniae from an individual in the community with asymptomatic bacteriuria.

BACKGROUND: The heteroresistance of polymyxin B, a last-resort antibiotic used to treat many serious bacterial infections, may lead to antibiotic treatment failure. However, polymyxin B-heteroresistant isolates are rare in individuals living in the community. We report a polymyxin B-heteroresistant hypervirulent Klebsiella pneumoniae (hvKP) isolate from an individual in the community with asymptomatic bacteriuria. RESULTS: The NYTJ35 isolate had multiple virulence genes that encoded a mucoid phenotype regulator (rmpA), aerobactin (iucABCD-iutA), salmochelin (iroBCDN), yersiniabactin (irp1-2 and ybtAEPQSTUX), and a truncated rmpA2. Infection of galleria mellonella larvae indicated the isolate was hypervirulent. Antimicrobial susceptibility testing showed it was susceptible to all tested antibiotics except polymyxin B. The proportion of surviving bacteria was 1.2 × 10- 7 based on the population analysis profile (PAP) method, suggesting the presence of polymyxin B heteroresistance. The isolate was not hypermucoviscous, but it was a strong biofilm producer. It had capsular serotype K1 and belonged to sequence type 23 (ST23). The isolate also had the D150G substitution in phoQ, which is known to confer polymyxin B resistance. CONCLUSIONS: We identified the co-occurrence of hypervirulence and polymyxin B heteroresistance in a K. pneumoniae isolate from an individual with asymptomatic bacteriuria. We suggest the use of increased screening for hvKP in individuals living in the community.

Emergence of High Prevalence of Extended-Spectrum Beta-Lactamase and Carbapenemase-Producing Enterobacteriaceae Species among Patients in Northwestern Ethiopia Region.

BACKGROUND: World Health Organization identified some Enterobacteriaceae as superbugs because of their high production and spread of extended-spectrum beta-lactamases (ESBL) and carbapenemases. Moreover, their resistance against commonly prescribed antibiotics left few choices of drugs to treat infection. This study is aimed at determining the magnitude of ESBL and carbapenemase-producing Enterobacteriaceae pathogens and their antimicrobial resistance pattern. MATERIALS AND METHODS: A hospital-based cross-sectional study was carried out from February to April 2019 in the Northwestern Ethiopia region. A total of 384 patients presumptive for bacterial infections were conveniently enrolled in the study. Specimens were collected and processed following standard bacteriological procedures. Drug susceptibility tests were performed using disk diffusion technique. ESBL and carbapenemase enzymes were tested by double disk diffusion and modified carbapenem inhibition methods, respectively. The data obtained were analyzed using SPSS version 22 software, and descriptive statistics were summarized in tables and graphs. RESULTS: Out of 384 clinical specimens processed 100 (26%) were culture positive for Enterobacteriaceae. The proportion of Enterobacteriaceae infection was relatively higher among in-patients 86 (32.6%) than out-patients 14 (11.7%). Overall, Escherichia coli 35 (9.1%) was the leading isolate followed by Klebsiella pneumoniae 31 (8.1%). Klebsiella pneumoniae 15 (15.6%) was the most frequent isolate from bloodstream infection and is the leading isolate from intensive care unit patients 15 (38.3%). Overall, 44 (44%) of Enterobacteriaceae were extended-spectrum beta-lactamase producers. Among them, Citrobacter spp. was the leading one 4 (80%) followed by Enterobacter cloacae 6 (60%) and K. pneumoniae 18 (58.1%). Furthermore, 6 (6%) of Enterobacteriaceae were carbapenemase-producers, in which 5 (50%) of E. cloacae and 3 (9.7%) of K. pneumoniae had highest percentage. Conclusions. ESBL and carbapenemase-producing isolates of Enterobacteriaceae are alarmingly spreading in the study area. Thus, improving the infection prevention strategy and further screening at the national level is recommended to develop the optimal use of antibiotics.

Methodology Establishment and Application of VITEK Mass Spectrometry to Detect Carbapenemase-Producing Klebsiella pneumoniae.

The ability of VITEK mass spectrometry (MS) in detection of bacterial resistance is currently under exploration and evaluation. In this study, we developed and validated a VITEK MS method to rapidly test carbapenemase-producing Klebsiella pneumoniae (CPKP). Solvents, antibiotic concentrations, crystal conditions and times, centrifugation speeds, and other factors were optimized to design a rapid sample pretreatment process for CPKP detection by VITEK MS. The related parameters of the mass spectrum were adjusted on the instrument to establish an CPKP detection mode. 133 clinically isolated strains of CPKP in the microbiology laboratory at the Shenzhen People's Hospital from 2004 to 2017 were selected for accuracy evaluation. The fresh suspected strains from the microbiology laboratory in 2020 were used to complete the clinical verification. Two antibiotics, meropenem (MEM) and imipenem (IPM), were used as substrates. These two substrates were incubated with suspected CPKP, and the results were obtained by VITEK MS detection. Using this method, different types of CPKP showed different detection results and all the CPKP strains producing KPC-2 and IMP-4 carbapenemase were detected by VITEK MS. Thus, VITEK MS can be used for rapid detection of CPKP, especially for some common types of CPKP. This method provides high accuracy and speed of detection. Combined with its cost advantages, it can be intensely valuable in clinical microbiology laboratories after the standard operating procedures are determined.

High Prevalence of Klebsiella pneumoniae in European Food Products: a Multicentric Study Comparing Culture and Molecular Detection Methods.

The Klebsiella pneumoniae species complex (KpSC) is a leading cause of multidrug-resistant human infections. To better understand the potential contribution of food as a vehicle of KpSC, we conducted a multicentric study to define an optimal culture method for its recovery from food matrices and to characterize food isolates phenotypically and genotypically. Chicken meat (n = 160) and salad (n = 145) samples were collected in five European countries and screened for the presence of KpSC using culture-based and zur-khe intergenic region (ZKIR) quantitative PCR (qPCR) methods. Enrichment using buffered peptone water followed by streaking on Simmons citrate agar with inositol (44°C for 48 h) was defined as the most suitable selective culture method for KpSC recovery. A high prevalence of KpSC was found in chicken meat (60% and 52% by ZKIR qPCR and the culture approach, respectively) and salad (30% and 21%, respectively) samples. Genomic analyses revealed high genetic diversity with the dominance of phylogroups Kp1 (91%) and Kp3 (6%). A total of 82% of isolates presented a natural antimicrobial susceptibility phenotype and genotype, with only four CTX-M-15-producing isolates detected. Notably, identical genotypes were found across samples-same food type and same country (15 cases), different food types and same country (1), and same food type and two countries (1)-suggesting high rates of transmission of KpSC within the food sector. Our study provides a novel isolation strategy for KpSC from food matrices and reinforces the view of food as a potential source of KpSC colonization in humans. IMPORTANCE Bacteria of the Klebsiella pneumoniae species complex (KpSC) are ubiquitous, and K. pneumoniae is a leading cause of antibiotic-resistant infections in humans. Despite the urgent public health threat represented by K. pneumoniae, there is a lack of knowledge of the contribution of food sources to colonization and subsequent infection in humans. This is partly due to the absence of standardized methods for characterizing the presence of KpSC in food matrices. Our multicentric study provides and implements a novel isolation strategy for KpSC from food matrices and shows that KpSC members are highly prevalent in salads and chicken meat, reinforcing the view of food as a potential source of KpSC colonization in humans. Despite the large genetic diversity and the low levels of resistance detected, the occurrence of identical genotypes across samples suggests high rates of transmission of KpSC within the food sector, which need to be further explored to define possible control strategies.

Factors influencing environmental sampling recovery of healthcare pathogens from non-porous surfaces with cellulose sponges.

Results from sampling healthcare surfaces for pathogens are difficult to interpret without understanding the factors that influence pathogen detection. We investigated the recovery of four healthcare-associated pathogens from three common surface materials, and how a body fluid simulant (artificial test soil, ATS), deposition method, and contamination levels influence the percent of organisms recovered (%R). Known quantities of carbapenemase-producing KPC+ Klebsiella pneumoniae (KPC), Acinetobacter baumannii, vancomycin-resistant Enterococcus faecalis, and Clostridioides difficile spores (CD) were suspended in Butterfield's buffer or ATS, deposited on 323cm2 steel, plastic, and laminate surfaces, allowed to dry 1h, then sampled with a cellulose sponge wipe. Bacteria were eluted, cultured, CFU counted and %R determined relative to the inoculum. The %R varied by organism, from <1% (KPC) to almost 60% (CD) and was more dependent upon the organism's characteristics and presence of ATS than on surface type. KPC persistence as determined by culture also declined by >1 log10 within the 60 min drying time. For all organisms, the %R was significantly greater if suspended in ATS than if suspended in Butterfield's buffer (p<0.05), and for most organisms the %R was not significantly different when sampled from any of the three surfaces. Organisms deposited in multiple droplets were recovered at equal or higher %R than if spread evenly on the surface. This work assists in interpreting data collected while investigating a healthcare infection outbreak or while conducting infection intervention studies.

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.

Combination of pre-adapted bacteriophage therapy and antibiotics for treatment of fracture-related infection due to pandrug-resistant Klebsiella pneumoniae.

A 30-year-old bombing victim with a fracture-related pandrug-resistant Klebsiella pneumoniae infection after long-term (>700 days) antibiotic therapy is treated with a pre-adapted bacteriophage along with meropenem and colistin, followed by ceftazidime/avibactam. This salvage therapy results in objective clinical, microbiological and radiological improvement of the patient's wounds and overall condition. In support, the bacteriophage and antibiotic combination is highly effective against the patient's K. pneumoniae strain in vitro, in 7-day mature biofilms and in suspensions.

Omic insights into various ceftazidime-avibactam-resistant Klebsiella pneumoniae isolates from two southern Italian regions.

Ceftazidime-avibactam (CZA) is one of the best therapeutic options available for infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria. However, sporadic reports of CZA-resistant strains have been rapidly increasing in patients. Herein, we provide detailed case reports of the emergence of ceftazidime-avibactam resistance to identify their resistome and virulome using genomic molecular approaches. Sixteen isolates were collected from 13 patients at three hospitals in Catania and Catanzaro (Italy) between 2020-2021. Antimicrobial susceptibility was determined by broth microdiluition. The samples included in study were analyzed for resistome, virulome and Sequence Type (ST) using Whole Genome Sequencing (WGS). All strains were resistant to ceftazidime/avibactam, ciprofloxacin, extended-spectrum cephalosporins and aztreonam, 13/16 to meropenem, 8/16 to colistin and 7/16 to fosfomycin; 15/16 were susceptible to meropenem/vaborbactam; all strains were susceptible to cefiderocol. Molecular analysis showed circulation of three major clones: ST101, ST307 and ST512. In 10/16 strains, we found a bla KPC-3 gene; in 6/16 strains, four different bla KPC variants (bla KPC28-31-34-50) were detected. A plethora of other beta-lactam genes (bla SHV28-45-55-100-106-187-205-212, bla OXA1-9-48, bla TEM-181 and bla CTX-M-15) was observed; bla OXA-9 was found in ST307 and ST512, instead bla OXA48 in one out four ST101 strains. With regard to membrane permeability, ompK35 and ompK36 harbored frameshift mutations in 15/16 strains; analysis of ompK37 gene revealed that all strains harbored a non-functional protein and carry wild-type PBP3. There is an urgent need to characterize the mechanisms underlying carbapenem resistance and the intrinsic bacterial factors that facilitate the rapid emergence of resistance. Furthermore, it is becoming increasingly important to explore feasible methods for accurate detection of different KPC enzymes.

Rapid detection of KPC-producing Klebsiella pneumoniae in China based on MALDI-TOF MS.

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) represent a serious threat to public health and their timely detection is essential for patient management and the prevention of nosocomial infections. Here, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to rapidly identify dominant KPC-Kp in China, by using an automated detection of a KPC-specific peak (at 4521 m/z) by a genetic algorithm using ClinProTools software. Whole-genome sequencing (WGS) was used to understand the genetic environment of the blaKPC-2 gene. In this study, we analyzed 235 K. pneumoniae Chinese clinical isolates, of which 175 (93 KPC-positive isolates and 82 KPC-negative isolates) isolates were used to build a model to select a KPC-specific peak, and another 60 isolates for external validation. In addition, all the spectra were visually inspected by the FlexAnalysis software to evaluate the accuracy of the automated detection. The results showed a 4521 m/z peak found in all blaKPC-2-positive isolates but absent in blaKPC-2-negative isolates. Interestingly, all KPC-Kp belonged to ST11, the dominant clone in China. WGS analysis of a representative isolate showed that the genetic environment of KPC-2 was IS26-ISKpn27-blaKPC-2-ΔISKpn6-Tn1721, similar to the KPC-2 genetic environment of ST11 KPC-Kp previously reported in China. Therefore, the 4521 m/z peak is closely related to ST11 KPC-Kp. In summary, we used MALDI-TOF MS to quickly detect KPC-Kp in the process of routine bacterial identification without increasing costs or requiring further knowledge, which has broad application prospects in drug resistance analysis and infection control.

Replicative transposition contributes to the evolution and dissemination of KPC-2-producing plasmid in Enterobacterales.

ABSTRACTKlebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales are prevalent worldwide and pose an alarming threat to public health. The incidence and transmission of blaKPC-2 gene via horizontal gene transfer (e.g. transposition) have been well documented. However, the dynamics of transposon structure bearing blaKPC-2 and their exact effects on the evolution and dissemination of blaKPC-2 gene are not well characterized. Here, we collected all 161 carbapenem-resistant Enterobacterales (CRE) isolates during the early stage of CRE pandemic. We observed that the prevalence of KPC-2-producing Enterobacterales was mediated by multiple species and sequence types (STs), and that blaKPC-2 gene was located on three diverse variants of Tn1721 in multi-drug resistance (MDR) region of plasmid. Notably, the outbreak of KPC-2-producing plasmid is correlated with the dynamics of transposon structure. Furthermore, we experimentally demonstrated that replicative transposition of Tn1721 and IS26 promotes horizontal transfer of blaKPC-2 and the evolution of KPC-2-producing plasmid. The Tn1721 variants appearing concurrently with the peak of an epidemic (A2- and B-type) showed higher transposition frequencies and a certain superior ability to propagation. Overall, our work suggests replicative transposition contributes to the evolution and transmission of KPC-2-producing plasmid and highlights its important role in the inter- and intra-species dissemination of blaKPC-2 gene in Enterobacterales.