Diagnostic Performance of BD Phoenix CPO Detect Panels for Detection and Classification of Carbapenemase-Producing Gram-Negative Bacteria.

BD Phoenix CPO Detect panels can identify and classify carbapenemase-producing organisms (CPOs) simultaneously with antimicrobial susceptibility testing (AST) for Gram-negative bacteria. Detection and classification of carbapenemase producers were performed using the BD Phoenix CPO Detect panels NMIC/ID-441 for Enterobacterales, NMIC/ID-442 for nonfermenting bacteria, and NMIC-440 for both. The results were compared with those obtained using comparator methods. A total of 133 strains (32 Klebsiella pneumoniae, 37 Enterobacter cloacae complex, 33 Pseudomonas aeruginosa, and 31 Acinetobacter baumannii complex strains), including 60 carbapenemase producers (54 imipenemases [IMPs] and 6 OXA type), were analyzed. Using panels NMIC-440 and NMIC/ID-441 or NMIC/ID-442, all 54 IMP producers were accurately identified as CPOs (positive percent agreement [PPA], 100.0%; 54/54). Among the 54 IMP producers identified as CPOs using panels NMIC-440 and NMIC/ID-441, 12 and 14 Enterobacterales were not resistant to carbapenem, respectively. Among all 54 IMP producers, 48 (88.9%; 48/54) were correctly classified as Ambler class B using panel NMIC-440. Using panels NMIC-440 and NMIC/ID-442, all four OXA-23-like carbapenemase-producing A. baumannii complex strains (100.0%, 4/4) were correctly identified as CPOs, and three (75.0%, 3/4) were precisely classified as class D using panel NMIC-440. Both carbapenemase producers harboring the blaISAba1-OXA-51-like gene were incorrectly identified as non-CPOs using panels NMIC-440 and NMIC/ID-442. For detecting carbapenemase producers, the overall PPA and negative percent agreement (NPA) between panel NMIC-440 and the comparator methods were 96.7% (58/60) and 71.2% (52/73), respectively, and the PPA and NPA between panels NMIC/ID-441 or NMIC/ID-442 and the comparator methods were 96.7% (58/60) and 74.0% (54/73), respectively. BD Phoenix CPO Detect panels can successfully screen carbapenemase producers, particularly IMP producers, regardless of the presence of carbapenem resistance and can be beneficial in routine AST workflows. IMPORTANCE Simple and efficient screening methods of detecting carbapenemase producers are required. BD Phoenix CPO Detect panels effectively screened carbapenemase producers, particularly IMP producers, with a high overall PPA. As the panels enable automatic screening for carbapenemase producers simultaneously with AST, the workflow from AST to confirmatory testing for carbapenemase production can be shortened. In addition, because carbapenem resistance varies among carbapenemase producers, the BD Phoenix CPO Detect panels, which can screen carbapenemase producers regardless of carbapenem susceptibility, can contribute to the accurate detection of carbapenemase producers. Our results report that these panels can help streamline the AST workflow before confirmatory testing for carbapenemase production in routine microbiological tests.

Rising incidence of carbapenem resistant isolates: an Argentinian hospital's experience. More trouble in the aftermath of the COVID-19 pandemic / Incidencia creciente de aislamientos resistentes a carbapenémicos: la experiencia de un hospital argentino. Más problemas tras la pandemia de COVID-1

Introduction: During COVID-19 outbreaks, disproportionate use of antibiotics, high Intensive Care Units burden and longer in-hospital stays may have aggravated the emergency posed by carbapenem-resistant isolates. Therefore, we set out to determine whether the incidence of carbapenem-resistant isolates rose in a tertiary care center in Santa Fe, Argentina during the period with active cases of COVID-19. Material and methods: In this retrospectively designed analytic epidemiologic study, two periods were defined: Period 1 (without active cases of COVID-19) from September 2019 to August 2020 and Period 2 (starting at the onset of the first wave of COVID-19 in this Institution) from September 2020 to June 2021. All clinically relevant microbiological samples taken during these periods in the Internal Medicine, Surgical and Intensive Care Unit wards were included. The primary analysis of interest was the differential incidence between the two periods, overall and in the Intensive Care Units wards in particular. Results: 9,135 hospitalizations, 50,145 patient-days of analysis. 7,285 clinical samples were taken, with an overall positivity for carbapenem-resistant isolates of 12.1% (n=883). Overall carbapenem-resistant isolates incidence during Period 2 was 2.5 times higher than in Period 1 (2.52 vs 0.955/100 patient-days, p<0.001). Intensive Care Units' carbapenem-resistant isolates incidence raised from 6.78 to 8.69/100 patient-days in Period 2 (p=0.006). Conclusions: We found alarming rates of carbapenem-resistant isolates in our center, 2.5 times higher in the period following the first wave of COVID-19. This rise was due to a higher amount of clinically relevant microbiological samples taken and to a higher carbapenem resistance among Enterobacteria and non-fermentative Gram-negative bacilli. To our knowledge, this is one of the few Latin-American reports on the effect of the COVID-19 pandemic on carbapenem-resistant isolates incidence (AU)

Introducción: Durante los brotes de COVID-19, el uso desproporcionado de antibióticos, la alta carga de las Unidades de Cuidados Intensivos y las estancias hospitalarias más prolongadas pueden haber agravado la emergencia planteada por los aislados resistentes a carbapenémicos. Por lo tanto, nos propusimos determinar si la incidencia de aislamientos resistentes a carbapenémicos aumentó en un centro de tercer nivel de atención en Santa Fe, Argentina, durante el período con casos activos de COVID-19. Material y métodos: En este estudio epidemiológico analítico de diseño retrospectivo se definieron dos periodos: Periodo 1 (sin casos activos de COVID-19) de septiembre de 2019 a agosto de 2020, y Periodo 2 (a partir del inicio de la primera ola de COVID-19. en esta Institución) desde septiembre de 2020 hasta junio de 2021. Se incluyeron todas las muestras microbiológicas clínicamente relevantes tomadas durante estos períodos en las salas de Medicina Interna, Quirúrgica y Unidad de Cuidados Intensivos. El principal análisis de interés fue la incidencia diferencial entre los dos períodos, en general y en las salas de las Unidades de Cuidados Intensivos en particular. Resultados: 9.135 hospitalizaciones, 50.145 pacientes-día de análisis. Se tomaron 7.285 muestras clínicas, con una positividad global para aislados resistentes a carbapenémicos del 12,1% (n=883). La incidencia general de aislamientos resistentes a carbapenémicos durante el Período 2 fue 2,5 veces mayor que en el Período 1 (2,52 frente a 0,955/100 pacientes-día, p<0,001). La incidencia de aislamientos resistentes a carbapenémicos en Unidades de Cuidados Intensivos aumentó de 6,78 a 8,69/100 pacientes-día en el Período 2 (p=0,006). Conclusiones: Encontramos tasas alarmantes de aislamientos resistentes a carbapenémicos en nuestro centro, 2,5 veces mayores en el período posterior a la primera ola de COVID-19 (AU)

Rapid Detection of Carbapenemase-producing Enterobacteriaceae From Blood Culture Bottles of Known CPE Carriers: Real-world Experience.

We used a rapid antigen test for the detection of carbapenemases directly from positive blood culture bottles of pediatric hemato-oncologic patients, known carriers of carbapenemase-producing enterobacteriaceae. Resistance mechanism was detected within 15 minutes of observing Gram-negative bacilli from a positive bottle, leading to treatment modification. This simple-to-use, inexpensive assay shortens the interval between empiric to tailored antimicrobial therapy.

Comparing mortality in patients with carbapenemase-producing carbapenem resistant Enterobacterales and non-carbapenemase-producing carbapenem resistant Enterobacterales bacteremia.

Carbapenem-resistant Enterobacterales (CRE) are classified as either carbapenemase-producing CRE (CP-CRE) or non-carbapenemase-producing CRE (non-CP-CRE) based on their mechanism of carbapenem resistance. Few studies have compared outcomes associated with each type of infection. We attempted to determine if either CRE subset is associated with increased mortality. We performed a retrospective observational study to collect demographic, clinical and outcomes data to compare patients with CP-CRE and non-CP-CRE bacteremia. Of 146 cases analyzed, 88/146 (60%) were CP-CRE and 58/146 (40%) were non-CP-CRE. Patients with CP-CRE bacteremia were less likely to receive active empiric or targeted antibiotic therapy. Non-CP-CRE bacteremia was associated with a 2.4 times higher hazard of death at 30 days after bacteremia onset compared to CP-CRE (HR, 2.4; 95% CI, 1.2, 4.6). Patients with non-CP-CRE bacteremia had a higher hazard of death at 30 days after bacteremia onset compared to those with CP-CRE bacteremia.

Bacterial characteristics of carbapenem-resistant Enterobacteriaceae (CRE) colonized strains and their correlation with subsequent infection.

BACKGROUND: Searching the risk factors for carbapenem-resistant Enterobacteriaceae (CRE) infection is important in clinical practice. In the present study, we aim to investigate bacterial characteristics of colonizing strains and their correlation with subsequent CRE infection. METHODS: Between May 2018 and January 2019, patients hospitalized in the department of haematology and intensive care unit (ICU) were screened for CRE by rectal swabs and monitored for the outcome of infection. We identified the species and carbapenemase-encoding genes of colonizing strains and performed antimicrobial susceptibility tests and multilocus sequence typing (MLST). Risk factors for subsequent CRE infections were ascertained by univariate and multivariable analysis. RESULTS: We collected a total of 219 colonizing strains from 153 patients. Klebsiella pneumoniae was the most abundant species, and MLST analysis showed rich diversity. K. pneumoniae carbapenemase (KPC) was predominant in the infection group (72.4%). In the non-infection group, 35.4% of strains were non-carbapenemase-producing CRE (NCP-CRE), and New Delhi metallo-ß-lactamase (NDM) was predominant (42.2%). The rate of high-level carbapenem resistance (minimum inhibitory concentration [MIC] ≥ 64 mg/L for meropenem and ertapenem, ≥ 32 mg/L for imipenem) was remarkably higher in the infection group than in the non-infection group (P <  0.001). Univariate analysis showed that K. pneumoniae, high-level carbapenem resistance, CP-CRE and KPC-CRE were infection risk factors after CRE colonization. On multivariable analysis with different carbapenemase dichotomizations, KPC-CRE (adjusted odds ratio [aOR], 4.507; 95% confidence interval [CI], 1.339-15.171; P = 0.015) or imipenem MIC ≥ 32 mg/L (aOR, 9.515; 95% CI, 1.617-55.977; P = 0.013) were respectively identified as independent risk factors for subsequent infection. CONCLUSIONS: Patients colonized with KPC-CRE or strains with an imipenem MIC ≥ 32 mg/L were at particularly high risk of subsequent CRE infections during their hospital stay.

Characterization of a carbapenem-resistant Citrobacter amalonaticus coharbouring bla IMP-4 and qnrs1 genes.

Introduction. Members of the genus Citrobacter are facultative anaerobic Gram-negative bacilli belonging to the Enterobacterales [Janda J Clin Microbiol 1994; 32(8):1850-1854; Arens Clin Microbiol Infect 1997;3(1):53-57]. Formerly, Citrobacter species were occasionally reported as nosocomial pathogens with low virulence [Pepperell Antimicrob Agents Chemother 2002;46(11):3555-60]. Now, they are consistently reported to cause nosocomial infections of the urinary tract, respiratory tract, bone, peritoneum, endocardium, meninges, intestines, bloodstream and central nervous system. Among Citrobacter species, the most common isolates are C. koseri and C. freundii, while C. amalonaticus has seldom been isolated [Janda J Clin Microbiol 1994; 32(8):1850-1854; Marak Infect Dis (Lond) 2017;49(7):532-9]. Further, Citrobacter spp. are usually susceptible to carbapenems, aminoglycosides, tetracyclines and colistin [Marak Infect Dis (Lond) 2017;49(7):532-9].Hypothesis/Gap Statement. As C. amalonaticus is rare, only one clinical isolate, coharbouring carbapenem resistance gene bla IMP-4 and quinolone resistance gene qnrs1, has been reported.Aim. To characterize a carbapenem-resistant C. amalonaticus strain from PR China coharbouring bla IMP-4 and qnrs1.Methodology. Three hundred and forty nonrepetitive carbapenem-resistant Enterobacterales (CRE) strains were collected during 2011-2018. A carbapenem-resistant C. amalonaticus strain was detected and confirmed using a VITEK mass spectrometry-based microbial identification system and 16S rRNA sequencing. Minimum inhibitory concentrations (MICs) for clinical antimicrobials were obtained by the broth microdilution method. Whole-genome sequencing (WGS) was performed for antibiotic resistance gene analysis, and a phylogenetic tree of C. amalonaticus strains was constructed using the Bacterial Pan Genome Analysis (BPGA) tool. The transferability of the resistance plasmid was verified by conjugal transfer.Results. A rare carbapenem-resistant C. amalonaticus strain (CA71) was recovered from a patient with cerebral obstruction and the sequences of 16S rRNA gene shared more than 99 % similarity with C. amalonaticus CITRO86, FDAARGOS 165. CA71 is resistant to ß-lactam, quinolone and aminoglycoside antibiotics, and even imipenem and meropenem (MICs of 2 and 4 mg l-1 respectively), and is only sensitive to polymyxin B and tigecycline. Six antibiotic resistance genes were detected via WGS, including the ß-lactam genes bla IMP-4, bla CTX-M-18 and bla Sed1, the quinolone gene qnrs1, and the aminoglycoside genes AAC(3)-VIIIa, AadA24. Interestingly, bla IMP-4 and qnrs1 coexist on an IncN1-type plasmid (pCA71-IMP) and successfully transferred to Escherichia coli J53 via conjugal transfer. Phylogenetic analysis showed that CA71 is most similar to C. amalonaticus strain CJ25 and belongs to the same evolutionary cluster along with seven other strains.Conclusion. To the best of our knowledge, this is the first report of a carbapenem-resistant C. amalonaticus isolate coharbouring bla IMP-4 and qnrs1.

Molecular analysis of clinical isolates of ceftazidime-avibactam-resistant Klebsiella pneumoniae.

OBJECTIVES: To analyse the strains collected during a 1-year survey of ceftazidime-avibactam-resistant KPC-producing Klebsiella pneumoniae, in order to investigate the molecular mechanisms potentially responsible for their resistant phenotype. METHODS: Clinical KPC-producing K. pneumoniae isolates were collected from 31 patients in six different hospitals in Rome. For eight of the patients, an additional strain grown before the start of treatment was also available, bringing the total of isolates studied to 39. Antimicrobial susceptibility was determined by automated system, broth microdiluition and E-test as appropriate. In silico analysis of acquired resistance genes was achieved by whole-genome sequencing, while multilocus sequence typing and core genome multilocus sequence typing were employed for molecular typing. Mutations associated with ceftazidime-avibactam resistance were identified by Sanger sequencing of the blaKPC gene. Possible mutations in OmpK35 and OmpK36 outer membrane proteins were also investigated. RESULTS: Molecular analyses highlighted the circulation of the ST512, 101 and 307 high-risk clones; 26 of the 31 patients carried a mutated KPC variant, five had a wild-type KPC-3. Among the KPC variants detected, 11 were different mutations within the blaKPC-3 gene, four of which were novel mutational changes. CONCLUSIONS: Different mutations including single amino-acid substitutions, insertions or deletions within the blaKPC gene were found in 26/31 ceftazidime-avibactam-resistant KPC-producing K. pneumoniae strains belonging to high-risk clones circulating in Italy. Of note, in 14/31 cases the isolates displayed resistance to both ceftazidime-avibactam and carbapenems, raising concerns for the possible selection of a multidrug-resistant phenotype.

Molecular characterization of carbapenem-resistant Enterobacteriaceae and emergence of tigecycline non-susceptible strains in the Henan province in China: a multicentrer study.

Introduction. Carbapenem-resistant Enterobacteriaceae (CRE) have been responsible for nosocomial outbreaks worldwide and have become endemic in several countries.Hypothesis/Gap Statement. To better understand the epidemiological trends and characteristics of CRE in the Henan province.Aim. We assessed the molecular epidemiological characteristics of 305 CRE strains isolated from patients in 19 secondary or tertiary hospitals in ten areas of the Henan province in China.Methodology. A total of 305 CRE isolates were subjected to multiple tests, including in vitro antimicrobial susceptibility testing, PCR for carbapenemase genes bla KPC, bla NDM, bla IMP, bla VIM, bla OXA-48-like. Tigecycline-resistant genes ramR, oqxR, acrR, tetA, rpsJ, tetX, tetM, tetL were analysed in five tigecycline non-susceptible carbapenem-resistant Klebsiella pneumoniae isolates (TNSCRKP). Additionally, multilocus sequence typing (MLST) was performed for carbapenem-resistant K. pneumoniae (CRKP).Results. The most common CRE species were K. pneumoniae (234, 77 %), Escherichia coli (36, 12 %) and Enterobacter cloacae (13, 4 %). All strains exhibited multi-drug resistance. Overall, 97 % (295/305) and 97 % (297/305) of the isolates were susceptible to polymyxin B and tigecycline, respectively. A total of 89 % (271/305) of the CRE isolates were carbapenemase gene-positive, including 70 % bla KPC, 13 % bla NDM, 6 % bla IMP, and 1 % combined bla KPC/bla NDM genes. K. pneumoniae carbapenemase (KPC) was the predominant carbapenemase in K. pneumoniae (87 %), whereas NDM and IMP were frequent in E. coli (53 %) and E. cloacae (69 %), respectively. Mutations in the ramR, tetA, and rpsJ genes were detected in five TNSCRKP. Moreover, 15 unique sequence types were detected, with ST11 (74 %), ST15 (9 %) and ST2237 (5 %) being dominant among K. pneumoniae strains.Conclusion. A high proportion of CRE strains were carbapenemase-positive, and five carbapenem-resistant K. pneumonia isolates were tigecycline non-susceptible, indicating a need for the ongoing surveillance of CRE and effective measures for the prevention of CRE infections.

Performance of the BD Phoenix CPO detect assay for detection and classification of carbapenemase-producing organisms.

Increasing worldwide, prevalence of carbapenem-resistant gram-negative bacteria demands urgent a need for rapid detection and accurate identification of carbapenemases. The BD Phoenix CPO detect (PCD) assay possesses an in-built capacity for parallel susceptibility testing and detection of carbapenemases. Here, the ability of the assay to detect and classify carbapenemase production was tested in a collection of carbapenem-resistant Enterobacterales and non-fermentative gram-negative rods. The ability of the PCD assay to detect and classify carbapenemases was investigated in a collection of 194 clinical, carbapenem-resistant isolates (Enterobacterales [n = 65]; non-fermentative gram-negative rods [n = 129]). AST results were compared to MICS determined by gradient diffusion to determine accuracy of the PCD assay. The accuracy of the PCD assay to detect carbapenemases was compared to the results of molecular isolate characterization using a LDT multiplex carbapenemase PCR assay. All 194 isolates classified as carbapenem-resistant by reference susceptibility testing were also classified correctly as CRO by the PCD assay. Performance analysis of the PCD assay to detect carbapenemase production revealed an overall sensitivity of 98.29% and specificity of 17.95% for the detection of carbapenemase production. For the classification of carbapenemases classes A, B, and D, the PCD correctly classified 79.17% Enterobacterales and 67.16% non-fermentative gram-negative rods. The PCD assay is a reliable tool for the detection of carbapenem resistance and allows for parallel analysis of carbapenemase production. However, while sensitivity is high, low specificity in carbapenemase detection and erroneous classification demands mandatory confirmation by alternative methods, especially in non-fermentative gram-negative bacteria.

Epigenomics, genomics, resistome, mobilome, virulome and evolutionary phylogenomics of carbapenem-resistant Klebsiella pneumoniae clinical strains.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) remains a major clinical pathogen and public health threat with few therapeutic options. The mobilome, resistome, methylome, virulome and phylogeography of CRKP in South Africa and globally were characterized. CRKP collected in 2018 were subjected to antimicrobial susceptibility testing, screening by multiplex PCR, genotyping by repetitive element palindromic (REP)-PCR, plasmid size, number, incompatibility and mobility analyses, and PacBio's SMRT sequencing (n=6). There were 56 multidrug-resistant CRKP, having blaOXA-48-like and blaNDM-1/7 carbapenemases on self-transmissible IncF, A/C, IncL/M and IncX3 plasmids endowed with prophages, traT, resistance islands, and type I and II restriction modification systems (RMS). Plasmids and clades detected in this study were respectively related to globally established/disseminated plasmids clades/clones, evincing transboundary horizontal and vertical dissemination. Reduced susceptibility to colistin occurred in 23 strains. Common clones included ST307, ST607, ST17, ST39 and ST3559. IncFIIk virulent plasmid replicon was present in 56 strains. Whole-genome sequencing of six strains revealed least 41 virulence genes, extensive ompK36 mutations, and four different K- and O-loci types: KL2, KL25, KL27, KL102, O1, O2, O4 and O5. Types I, II and III RMS, conferring m6A (GATC, GATGNNNNNNTTG, CAANNNNNNCATC motifs) and m4C (CCWGG) modifications on chromosomes and plasmids, were found. The nature of plasmid-mediated, clonal and multi-clonal dissemination of blaOXA-48-like and blaNDM-1 mirrors epidemiological trends observed for closely related plasmids and sequence types internationally. Worryingly, the presence of both blaOXA-48 and blaNDM-1 in the same isolates was observed. Plasmid-mediated transmission of RMS, virulome and prophages influence bacterial evolution, epidemiology, pathogenicity and resistance, threatening infection treatment. The influence of RMS on antimicrobial and bacteriophage therapy needs urgent investigation.

A method to quantify viable carbapenem resistant gram-negative bacteria in treated and untreated wastewater.

Carbapenem resistance, particularly in Enterobacteriaceae, is an urgent threat to public health worldwide. Wastewater treatment plants are a critical control point for the spread of antimicrobial resistance into the environment yet, due in part to the lack of appropriate methods, the occurrence, identification and removal of carbapenem resistant bacteria has not been well characterized in wastewater matrices. This project was designed to provide a method for quantification of viable carbapenem resistant (CR) gram-negative bacteria (GNB) in raw sewage and treated wastewater effluents. A two-step procedure using membrane filtration and selective media supplemented with each of four carbapenems (doripenem, meropenem, imipenem, and ertapenem) was established for the quantification of CR GNB in wastewater matrices. Carbapenemase production was also assessed on individual bacterial colonies using two separate methods. Vitek®2 antimicrobial susceptibility test and disk diffusion assays were used to verify results from the supplemented media test and provide taxonomic identification. Treated and untreated wastewater samples from secondary and tertiary-stage wastewater treatment plants were analyzed for CR bacteria using the supplemented media procedure. Over 98% of all isolates selected from the carbapenem-supplemented media were verified as CR GNB. Carbapenemase production was observed in 80% of these isolates and 88% were multidrug resistant. All Enterobacteriaceae isolates from the supplemented media were verified as CR and 97% tested positive for carbapenemase production. The highest concentrations of CR GNB in wastewater were observed using the ertapenem-supplemented media. Doripenem-supplemented media showed the greatest specificity and selectivity for carbapenemase-producing CRE. Overall, the cumulative CR GNB in wastewater were reduced by approximately three- and five-log10 by the secondary and tertiary-stage WWTPs, respectively. This study establishes a method for characterization of viable CR GNB in wastewater matrices and demonstrates that current wastewater treatment technologies effectively reduce CR bacteria, including CRE, in sewage.

Epidemiology and risk factors of rectal colonization of carbapenemase-producing Enterobacteriaceae among high-risk patients from ICU and HSCT wards in a university hospital.

BACKGROUND: Nosocomial carbapenemase-producing Enterobacterieceae (CPE) infections constitute a major global health concern and are associated with increased morbidity and mortality. Rectal colonization with CPE is a risk factor for bacterial translocation leading to subsequent endogenous CPE infections. This prospective observational study was aimed to investigate the prevalence and epidemiology of rectal colonization of CPE, the carbapenemase genotypes, and to identify the independent risk factors for the acquisition of CPE colonization in high-risk patients from ICU and HSCT wards in a university hospital in China. METHODS: In a prospective cohort study, 150 fecal samples from rectal swabs were consecutively obtained for inpatients from the intensive care unit (ICU) and hematopoietic stem cell transplantation (HSCT) wards from November 2018 to May 2019, and screening test for CPE was conducted by using prepared in-house trypsin soybean broth (TSB) selective media and MacConkey agar. Antimicrobial susceptibility was determined by the broth microdilution method and carbapenemase genes were characterized by both the GeneXpert Carba-R and PCR for blaKPC, blaNDM, blaIMP, blaVIM and blaOXA. Multi-locus sequence typing (MLST) was employed to characterize the genetic relationships among the carbapenemase-producing K. Pneumonia (CPKP) isolates. In order to further investigate the risk factors and clinical outcomes of CPE colonization, a prospective case-control study was also performed. RESULTS: Twenty-six suspected CPE strains, including 17 Klebsiella pneumoniae, 6 Escherichia coli, 1 Citrobacter freundii, 1 Enterobacter Kobe, and 1 Raoultella ornithinolytica, were identified in 25 non-duplicated rectal swab samples from 25 patients, with a carriage rate of 16.67% (25/150). Through GeneXpert Carba-R and subsequent PCR and sequencing, all the suspected CPE isolates were identified to be positive for the carbapenemase genes, of which 17 were blaKPC-carriers, and another 9 were blaNDM-producers. MLST designated all the CPKP isolates to be ST11 clone. Multivariate analysis indicated that urinary system diseases, operation of bronchoscopy, and combined use of antibiotics were independent risk factors for acquiring CPE colonization in high-risk patients from the ICU and HSCT wards. CONCLUSIONS: This study revealed a high prevalence of rectal CPE colonization in high-risk patients from ICU and HSCT wards, and a predominant colonization of the KPC-producing K. pneumoniae clone ST11. Stricter infection control measures are urgently needed to limit the dissemination of CPE strains, especially in patients who were afflicted by urinary system diseases, have underwent bronchoscopy, and were previously exposed to combined antibiotic use.

Successful control of the first carbapenem-resistant Klebsiella pneumoniae outbreak in a Chinese hospital 2017-2019.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is considered as a serious global threat. CRKPs occurred only sporadically in the Second Hospital of Shanxi Medical University. Our study aimed to investigate and control the first outbreak of CRKP in our hospital occurred between October 2017 and August 2019. METHODS: The antimicrobial stewardship (AMS) workers have been implemented control measures properly. Clinical and epidemiological data were retrospectively collected from medical records. Carbapenemase genes were detected by modified carbapenem inactivation method (mCIM) test and the EDTA-modified carbapenem inactivation method (eCIM) test. Resistance genes were identified by polymerase chain reaction (PCR) and sequencing. Genetic relatedness was studied by multilocus sequence typing (MLST). RESULTS: During the outbreak, 31 patients were infected with CRKP isolates. 20 (64.5%) patients were infected with KPC-2 and/or NDM-1 producing K. pneumoniae. Mostly MLST-sequence types belonged to ST11 (21/31). The outbreak was two major K. pneumoniae clusters present in epidemiologically linked patients. CONCLUSIONS: Setting up AMS workers is potentially a highly efficient strategy for the successful control of the outbreak. A multimodal and multidisciplinary infection control strategy proved to be crucial. The emergence of CRKP in our hospital emphasizes the importance of continuous monitoring of these isolates, which helps to limit the spread of CRKPs and improve the level of management.

Emergence of ß-lactamase- and carbapenemase- producing Enterobacteriaceae at integrated fish farms.

BACKGROUND: Epidemiological studies suggested that determinants for antibiotic resistance have originated in aquaculture. Recently, the integrated agriculture-aquaculture system has been implemented, where fish are raised in ponds that receive agriculture drainage water. The present study aims to investigate the occurrence of ß-lactamase and carbapenemase-producing Enterobacteriaceae in the integrated agriculture-aquaculture and the consequent public health implication. METHODS: Samples were collected from fish, fishpond water inlets, tap water, outlet water, and workers at sites of integrated agriculture-aquacultures. Samples were also taken from inhabitants of the aquaculture surrounding areas. All samples were cultured on MacConkey agar, the Enterobacteriaceae isolates were tested for susceptibility to cephalosporins and carbapenems, and screened for blaCTX-M-15, blaSHV, blaOXA-1, blaTEM, blaPER-1, blaKPC, blaOXA-48, and blaNDM. Strains having similar resistance phenotype and genotype were examined for the presence of Incompatible (Inc) plasmids. RESULTS: A major proportion of the Enterobacteriaceae isolates were resistant to cephalosporins and carbapenems. Among the 66 isolates from fish, 34 were resistant to both cephalosporin and carbapenem groups, 26 to carbapenems alone, and 4 to cephalosporins alone. Of the 15 isolates from fishpond water inlets, 8 showed resistance to both groups, 1 to carbapenems alone, and 5 to cephalosporins alone. Out of the 33 isolates from tap water, 17 were resistant to both groups, and 16 to cephalosporins alone. Similarly, of the 16 outlet water isolates, 10 were resistant to both groups, and 6 to cephalosporins alone. Furthermore, of the 30 examined workers, 15 carried Enterobacteriaceae resistant strains, 10 to both groups, and 5 to cephalosporins alone. Similar strains were isolated from the inhabitants of the aquaculture surrounding areas. Irrespective of source of samples, strains resistant to all examined antibiotics, carried predominantly the carbapenemase gene blaKPC either alone or with the ß-lactamase genes (blaCTX-M-15, blaSHV, blaTEM, and blaPER-1). The isolates from fish, water, and workers harboured a wide-range of multi-drug-resistance Inc. plasmids, which were similar among all isolates. CONCLUSION: The present findings suggest transmission of the resistance genes among Enterobacteriaceae strains from different sources. This reiterates the need for control strategies that focus on humans, animals, water, and sewage systems to solve the antibiotic resistance problem.

Molecular Epidemiology of Carbapenem-Resistant Enterobacterales Strains Isolated from Blood Cultures in Antalya, Turkey.

OBJECTIVE: The aim of this study was to investigate the prevalence of carbapenemase and CTX-M genes among 330 blood culture isolates of Enterobacterales with reduced susceptibility to at least 1 carbapenem, between 2010 and 2015. METHODS: BD Max CRE assay and in-house PCR were used to detect carbapenemase and CTX-M genes. RESULTS: At least 1 carbapenemase gene was detected among 113 (74.3%) of the 152 carbapenem resistant isolates. The OXA-48 (69.7%) was the most common carbapenemase followed by VIM, NDM and IMP, whereas no tested isolates were KPC-positive. Eighty-six isolates (56.6%) had CTX-M and 65 had both OXA-48 and CTX-M. Carbapenemase production in Enterobacterales was significantly increased in years (P < .05). CONCLUSION: Our study indicates that there is ongoing endemic circulation of the OXA-48 producing organism in our facility. It is noteworthy that more than half of the OXA-48 producing strains also produced CTX-M enzyme.

Characterization of carbapenem-resistant Klebsiella pneumoniae in a tertiary hospital in Fuzhou, China.

AIMS: The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains has led to increased mortality and morbidity rates. Tigecycline, a new class of broad-spectrum glycyl-tetracycline antibiotics, has been used to target multi- and pan-drug-resistant bacterial infections. This study aimed to assess the molecular characteristics of CRKP in a tertiary hospital, and its susceptibility to tigecycline, to create a reference for hospital infection control and clinical drug use. METHODS AND RESULTS: We retrieved patient clinical information and CRKP characterization from medical records and detected the MIC of tigecycline using the micro-broth dilution method. Multi-locus sequence typing was performed, and antibiotic resistance genes associated with CRKP were detected by qPCR. A total of 166 CRKP strains were detected in the sputum, urine and blood among intensive care unit patients (average age, 69·6 years). The most infrequently observed resistance genes were amikacin resistance genes, followed by tobramycin resistance genes. KPC-2, CTX-M9 and CTX-M1 were the most frequently detected resistance genes. CONCLUSIONS: No strain was resistant to tigecycline (MIC ≥ 8 µg ml-1 ). Twenty-four sequence types were identified, with ST11 being the most common type. SIGNIFICANCE AND IMPACT OF THE STUDY: Clinicians and infection control experts should be aware of CRKP prevalence to facilitate clinical treatment and improve nosocomial infection control.

Adaptive evolution of virulence and persistence in carbapenem-resistant Klebsiella pneumoniae.

Among the most urgent public health threats is the worldwide emergence of carbapenem-resistant Enterobacteriaceae1-4, which are resistant to the antibiotic class of 'last resort'. In the United States and Europe, carbapenem-resistant strains of the Klebsiella pneumoniae ST258 (ref. 5) sequence type are dominant, endemic6-8 and associated with high mortality6,9,10. We report the global evolution of pathogenicity in carbapenem-resistant K. pneumoniae, resulting in the repeated convergence of virulence and carbapenem resistance in the United States and Europe, dating back to as early as 2009. We demonstrate that K. pneumoniae can enhance its pathogenicity by adopting two opposing infection programs through easily acquired gain- and loss-of-function mutations. Single-nucleotide polymorphisms in the capsule biosynthesis gene wzc lead to hypercapsule production, which confers phagocytosis resistance, enhanced dissemination and increased mortality in animal models. In contrast, mutations disrupting capsule biosynthesis genes impair capsule production, which enhances epithelial cell invasion, in vitro biofilm formation and persistence in urinary tract infections. These two types of capsule mutants have emerged repeatedly and independently in Europe and the United States, with hypercapsule mutants associated with bloodstream infections and capsule-deficient mutants associated with urinary tract infections. In the latter case, drug-tolerant K. pneumoniae can persist to yield potentially untreatable, persistent infection.

Carbapenem-resistant Enterobacteriaceae in patients with bacteraemia at tertiary hospitals in South Africa, 2015 to 2018.

Enhanced surveillance for CREs was established at national sentinel sites in South Africa. We aimed to apply an epidemiological and microbiological approach to characterise CREs and to assess trends in antimicrobial resistance from patients admitted to tertiary academic hospitals. A retrospective analysis was conducted on patients of all ages with CRE bacteraemia admitted at any one of 12 tertiary academic hospitals in four provinces (Gauteng, KwaZulu-Natal, Western Cape and Free State) in South Africa. The study period was from July 2015 to December 2018. A case of CRE bacteraemia was defined as a patient admitted to one of the selected tertiary hospitals where any of the Enterobacteriaceae was isolated from a blood culture, and was resistant to the carbapenems (ertapenem, meropenem, imipenem and/or doripenem) or had a positive result for the Modified Hodge Test (MHT) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. A positive blood culture result obtained after 21 days of the last blood culture result was regarded as a new case. To distinguish hospital-acquired (HA) from the community-acquired (CA) bacteraemia, the following definitions were applied: the HA CRE bacteraemia was defined as a patient with CRE isolated from blood culture ≥ 72 h of hospital admission or with any prior healthcare contact, within 1 year prior to the current episode or referral from a healthcare facility where the patient was admitted before the current hospital. A case of the CA CRE bacteraemia was defined as a patient with CRE isolated from blood culture < 72 h of hospital admission and with no prior healthcare contact. The majority of carbapenem-resistant Enterobacteriaceae (CRE) (70%) were hospital-acquired (HA) with Klebsiella pneumoniae being the predominant species (78%). In-hospital mortality rate was 38%. The commonest carbapenemase genes were bla-OXA-48 (52%) and bla-NDM (34%). The high mortality rate related to bacteraemia with CRE and the fact that most were hospital-acquired infections highlights the need to control the spread of these drug-resistant bacteria. Replacement with OXA-48 is the striking finding from this surveillance analysis. Infection control and antibiotic stewardship play important roles in decreasing the spread of resistance.

Emergence of ST11-K47 and ST11-K64 hypervirulent carbapenem-resistant Klebsiella pneumoniae in bacterial liver abscesses from China: a molecular, biological, and epidemiological study.

Background: Multidrug-resistant bacteria, especially those with high virulence, are an emerging problem in clinical settings.Methods: We conducted a multicentre epidemiological and comparative genomic analysis on the evolution, virulence and antimicrobial resistance of carbapenem-resistant Enterobacteriaceae in patients with bacterial liver abscesses from 2012 to 2016.Results: A total of 477 bacterial isolates were collected. Enterobacteriaceae were the main pathogen (89.3%) with K. pneumoniae (52.4%) predominating followed by Escherichia coli (26.8%). All CRKps (3.2%) were of sequence type (ST) 11 and serotypes K47 or K64, and simultaneously possessed acquired blaKPC-2/blaKPC-5 and blaCTX-M-65 together with the multidrug transporter EmrE. Seven Hv-CRKps (five ST11-K47, two ST11-K64) were confirmed by bacteriological test, neutrophil killing assay and Galleria mellonella infection model. Genomic analysis indicated that the emergence of one ST11-K64 Hv-CRKp strain was related to the acquisition of rmpA/rmpA2 genes and siderophore gene clusters, while ST11-K47 Hv-CRKp lacked these traditional virulence genes. Further complete genome analysis of one ST11-K47 Hv-CRKp strain, R16, showed that it acquired a rare plasmid (pR16-Hv-CRKp1) carrying blaKPC-2, blaSHV-12, blaTEM-1, blaCTX-M-65, rmtB and a predicted virulence gene R16_5486 simultaneously.Conclusion: The emergence of the ST11-K47/K64 Hv-CRKps, which are simultaneously multidrug-resistant and hypervirulent, requires urgent control measures to be implemented.

The emergence of carbapenemase blaNDM genotype among carbapenem-resistant Enterobacteriaceae isolates from Egyptian cancer patients.

Carbapenem resistance among Enterobacteriaceae is a major concern that is increasingly reported worldwide. The objective of this study is to determine the incidence of carbapenem resistance as well as to investigate for carbapenemase-encoding genes among Enterobacteriaceae clinical isolates from cancer patients at different cancer institutes in Egypt. This determination was a cross-sectional study with a total of 135 clinical isolates collected over a period of 1 year. All isolates were sub-cultured on ChromID agar and subjected to phenotypic and molecular detection of carbapenemases. Most of the Enterobacteriaceae isolates were MDR with high resistance rates against tested antimicrobials. Overall, the results of PCR assays revealed that 89.62% (121/135) of isolates harbored one or more of the carbapenemase-encoding genes, while phenotypic assays revealed the production of carbapenemases in 68.88% (93/135) of isolates. BlastN analysis against the non-redundant genome sequences available in the GenBank database revealed that the blaNDM-1 gene was the most prevalent genotype of carbapenemases in 93/135 (68.88%), followed by blaOXA-48 44/135 (32.59%), blaOXA-23 42/135 (31.11%), and blaKPC-2 2/135 (1.48%). Klebsiella pneumoniae isolates harbored the highest number of carbapenemase-encoding genes 34/121 (28.09%). The high prevalence of carbapenemases and/or their encoding genes among MDR Enterobacteriaceae bacteria in Egypt is alarming, thus, the management of serious infections caused by Enterobacteriaceae, particularly in cancer patients will be challenging to clinicians. Carbapenemase blaNDM genotype is emerging in cancer healthcare settings in Egypt, which could be the cause of the current increase in carbapenemase-producing Enterobacteriaceae.