Emergence of Klebsiella pneumoniae co-producing NDM-type and OXA-181 carbapenemases

The emergence of carbapenemase-producing Enterobacteriaceae is a rapidly evolving threat worldwide. Here, we report the molecular characterization of two Klebsiella pneumoniae isolates carrying both bla_OXA-181 and bla_NDM-1 or bla_NDM-5 isolated from epidemiologically unrelated patients in Singapore. The bla_OXA-181 genes were found existing in different genetic environments.     

Klebsiella pneumoniae harbouring OXA-48 carbapenemase in a Libyan refugee in Italy

A carbapenem-resistant Klebsiella pneumoniae was isolated from a blood-culture of an inpatient from Libya, hospitalized in the intensive-care unit of Negrar Hospital, Italy. The clinical isolate carried the following β-lactamase genes, bla_TEM-1, bla_SHV-11, bla_OXA-1, bla_CTX-M-15 and bla_OXA-48, respectively. The bla_OXA-48 gene was inserted in the Tn1999.2 transposon type, carried on a conjugative, 60-kilobase plasmid, that presented an L/M backbone, hosted by a multidrug-resistant ST 101 K. pneumoniae strain. Our report highlights the international transfer of bla_OXA-48 gene and the importance of screening measures of multidrug-resistant Enterobacteriaceae.     

Phenotypic and Molecular Characteristics of Carbapenem-Resistant Enterobacteriaceae in a Health Care System in Los Angeles,California, from 2011 to 2013

Carbapenem-resistant Enterobacteriaceae (CRE) are a concern for health care in the United States but remain relatively uncommon in California. We describe the phenotype, clonality, and carbapenemase-encoding genes present in CRE isolated from patients at a Californian tertiary health care system. CRE for this study were identified by evaluating the antibiograms of Enterobacteriaceae isolated in the UCLA Health System from 2011 to 2013 for isolates that were not susceptible to meropenem and/or imipenem. The identification of these isolates was subsequently confirmed by matrix-associated laser desorption ionization–time of flight, and broth microdilution tests were repeated to confirm the CRE phenotype. Real-time PCR for bla_KPC, bla_SME, bla_IMP, bla_NDM-1, bla_VIM, and bla_OXA-48 was performed. Clonality was assessed by repetitive sequence-based PCR (repPCR) and multilocus sequence typing (MLST). Of 15,839 nonduplicate clinical Enterobacteriaceae isolates, 115 (0.73%) met the study definition for CRE. This number increased from 0.5% (44/8165) in the first half of the study to 0.9% (71/7674) in the second (P = 0.004). The most common CRE species were Klebsiella pneumoniae, Enterobacter aerogenes, and Escherichia coli. A carbapenemase-encoding gene was found in 81.7% (94/115) of CRE and included bla_KPC (78.3%), bla_NDM-1 (0.9%), and bla_SME (2.6%). The majority of bla_KPC genes were in K. pneumoniae isolates, which fell into 14 clonal groups on typing. bla_KPC was identified in more than one species of CRE cultured from the same patient in four cases. Three bla_SME-carrying Serratia marcescens isolates and one bla_NDM-1 carrying Providencia rettgeri isolate were detected. CRE are increasing in California, and carbapenemases, particularly KPC, are a common mechanism for carbapenem resistance in this region.     

Genomics of Klebsiella pneumoniae ST16 producing NDM-1, CTX-M-15, and OXA-232

Objectives

Genomic characterization of the internationally spread sequence type (ST) 16 carbapenem-resistant Klebsiella pneumoniae.

Molecular epidemiology and resistance patterns of blaOXA-48 Klebsiella pneumoniae and Escherichia coli: A nationwide multicenter study in Taiwan

BackgroundWe describe the molecular epidemiology and resistance patterns of bla_OXA-48 Klebsiella pneumoniae and Escherichia coli in Taiwan.MethodsIn this multicenter surveillance study from January 2012 to August 2015, the identified bla_OXA-48 Enterobacteriaceae isolates were subjected to antibiotics susceptibility testing. PCR method was used for detecting concomitant other beta-lactamases. Outer membrane porins were analyzed. Genetic relatedness and molecular epidemiology of the isolates were determined through pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmid incompatibility was determined using PCR-based replicon typing.ResultsForty-three bla_OXA-48 K. pneumoniae and two E. coli isolates were analyzed. The annual incidence of bla_OXA-48 K. pneumoniae isolates from 2012 to 2015 were 0%, 1.1%, 2.4%, and 7.6%, respectively. Forty-three (95.5%) of 45 isolates were non-susceptible to broad-spectrum beta-lactams (ceftriaxone, ceftazidime, cefepime, piperacillin/tazobactam), Forty-two (93.3%) of the 45 isolates showed resistance against all tested carbapenems (imipenem, meropenem, doripenem, and ertapenem). Molecular characterization revealed that they co-produced at least one extended-spectrum beta-lactamases or AmpC beta-lactamases, with at least one outer membrane porin loss. Thirty-eight (88.3%) of the 43 K. pneumoniae isolates belonged to ST11. PFGE analysis of 43 K. pneumoniae isolates revealed dissemination of multiple clones. Six of the 12 tested K. pneumoniae representatives of different pulso-types belonged to IncA/C.ConclusionConcomitant loss of porins and production of other beta-lactamases renders the bla_OXA-48-producing isolates in Taiwan a high-level carbapenem resistance and broad resistance against many beta-lactam antibiotics. Following dissemination of multiple clones of bla_OXA-48 K pneumoniae ST 11, a trend of increased bla_OXA-48 prevalence was noted.     

Unusual association of NDM-1 with KPC-2 and armA among Brazilian Enterobacteriaceae isolates

We report the microbiological characterization of four New Delhi metallo-β-lactamase-1 (bla _NDM-1)-producing Enterobacteriaceae isolated in Rio de Janeiro, Brazil. bla _NDM-1 was located on a conjugative plasmid and was associated with Klebsiella pneumoniae carbapenemase-2 (bla _KPC-2) or aminoglycoside-resistance methylase (armA), a 16S rRNA methylase not previously reported in Brazil, in two distinct strains of Enterobacter cloacae. Our results suggested that the introduction of bla _NDM-1 in Brazil has been accompanied by rapid spread, since our isolates showed no genetic relationship.     

Molecular characteristics of carbapenemase-producing Enterobacterales in the Netherlands; results of the 2014–2018 national laboratory surveillance

ObjectivesCarbapenem resistance mediated by mobile genetic elements has emerged worldwide and has become a major public health threat. To gain insight into the molecular epidemiology of carbapenem resistance in The Netherlands, Dutch medical microbiology laboratories are requested to submit suspected carbapenemase-producing Enterobacterales (CPE) to the National Institute for Public Health and the Environment as part of a national surveillance system.MethodsMeropenem MICs and species identification were confirmed by E-test and MALDI-TOF and carbapenemase production was assessed by the Carbapenem Inactivation Method. Of all submitted CPE, one species/carbapenemase gene combination per person per year was subjected to next-generation sequencing (NGS).ResultsIn total, 1838 unique isolates were received between 2014 and 2018, of which 892 were unique CPE isolates with NGS data available. The predominant CPE species were Klebsiella pneumoniae (n = 388, 43%), Escherichia coli (n = 264, 30%) and Enterobacter cloacae complex (n = 116, 13%). Various carbapenemase alleles of the same carbapenemase gene resulted in different susceptibilities to meropenem and this effect varied between species. Analyses of NGS data showed variation of prevalence of carbapenemase alleles over time with bla_OXA-48 being predominant (38%, 336/892), followed by bla_NDM-1 (16%, 145/892). For the first time in the Netherlands, bla_OXA-181, bla_OXA-232 and bla_VIM-4 were detected. The genetic background of K. pneumoniae and E. coli isolates was highly diverse.ConclusionsThe CPE population in the Netherlands is diverse, suggesting multiple introductions. The predominant carbapenemase alleles are bla_OXA-48 and bla_NDM-1. There was a clear association between species, carbapenemase allele and susceptibility to meropenem.     

Development of TaqMan real-time polymerase chain reaction for the detection of the newly emerging form of carbapenem resistance gene in clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii

Purpose: The newly emerging form of the so-called New Delhi Metallo-beta-lactamases (NDM-1) has been reported recently from patients worldwide and broadly thought as a potential source for the major global health problem. Thus, it is important to study the epidemiology of the so-called NDM-1 harbouring bacteria to prevent its further spread and to place effective control measures. The present study describes the use of the real-time polymerase chain reaction (PCR) assay for the detection of the bla_NDM-1 gene using TaqMan probes among clinical isolates. Materials and Methods: Clinical isolates of Escherichia coli (11 strains), Klebsiella pneumoniae (17 strains) and Acinetobacter baumannii (six strains) that were resistant to either of the carbapenems (meropenem or imipenem) were included in the study. The presence of carbapenemases in such strains was confirmed using the modified Hodge test. A real-time PCR assay was optimized for the detection of NDM-1 using a cloned synthetic gene fragment followed by testing of the clinical isolates. The findings were further confirmed using PCR and gene sequencing. Results: TaqMan probe assay displayed a good detection limit with analytical sensitivity of the assay up to 10 copies of bla_NDM-1 gene per reaction. The isolates of E. coli and K. pneumoniae revealed narrow range crossing point values (Cp values) between (12–17) cycles (mean Cp value 14), indicating number of bla_NDM-1 gene copies of 106–108. The wider range of Cp values (15–34) cycles with a higher mean Cp value (23.6) was observed in A. baumannii with number of bla_NDM-1 gene copies of 103–108. Conclusions: The study demonstrates that real-time PCR assay based on TaqMan chemistry is a useful technique for the detection of bla_NDM-1 harbouring clinical isolates of E. coli, K. pneumoniae and A. baumannii. The assay has great precision in measuring the number of bla_NDM-1 gene copies per specimen of DNA.     

Diversity of carbapenemases in clinical isolates of Enterobacteriaceae in Croatia—the results of a multicentre study

Since the first carbapenem-resistant Klebsiella pneumoniae strain was isolated in 2008, Enterobacteriaceae with reduced susceptibility to one or more carbapenems have emerged sporadically in different geographical regions in Croatia. These observations gave rise to a multicenter study on carbapenem resistance in Enterobacteriaceae from Croatia. Fifty-seven carbapenem-non-susceptible strains of Enterobacteriaceae were collected during 2011–2012 from four large hospital centres in Croatia. Overall, 36 strains produced VIM-1 β-lactamase, three produced NDM-1, and one produced KPC-2. A high degree of clonal relatedness was observed in Enterobacter cloacae and Citrobacter freundii strains, in contrast to K. pneumoniae strains. Bla_VIM genes were located within class1 integron which contained genes encoding resistance to aminoglycosides (aac_A4). The study found strong association between bla_VIM and qnr_B6 and between bla_NDM and qnr_A6 genes.     

Emergence of NDM-producing non-baumannii Acinetobacter spp. isolated from China

One hundred and thirty-six bla _OXA-51-negative strains were identified from 1,067 Acinetobacter calcoaceticus–A. baumannii complex (ACB complex) isolates, which were collected during October 2010 to March 2013 from 15 general hospitals in 10 cities throughout Zhejiang Province, China. Seven of the 136 bla _OXA-51-negative ACB complex isolates were New Delhi metallo-β-lactamase-1 (NDM-1)-positive, among which three were identified as A. nosocomialis and four were identified as A. pittii strains using 16S–23S rRNA gene intergenic spacer (ITS) sequencing and partial RNA polymerase β-subunit (rpoB) sequencing. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analysis showed that the seven NDM-positive isolates belonged to three clonal strains with three novel sequence types (STs). Polymerase chain reaction (PCR) assays and DNA sequence analysis of the carbapenemase and other β-lactamase genes indicated that all the isolates harbored the bla _NDM-1 gene, and that only one strain of A. nosocomialis isolates harbored both bla _NDM-1 and bla _OXA-23. All of them were positive for bla _ADC, from which three novel bla _ADC genes (designated as bla _ADC-69, bla _ADC-70, and bla _ADC-71) were detected for the first time. The presence of ISAba125 upstream of bla _NDM-1 was identified through genetic environment analysis. Carbapenem resistance can be transferred from A. nosocomialis and A. pittii to Escherichia coli EC600 by the conjugation experiment. Plasmid analysis, DNA hybridization, and extraction experiments indicated that bla _NDM-1 was located on a plasmid of approximately 50 kb. In conclusion, we characterized the dissemination of NDM-1-positive A. pittii strains in Zhejiang Province, China, and reported the NDM-producing A. nosocomialis for the first time.     

Dissemination of <Emphasis Type="Italic">bla</Emphasis><Subscript>OXA-370</Subscript> is mediated by IncX plasmids and the Tn6435 transposon

In Enterobacteriaceae, the bla_OXA-48-like genes have been identified on plasmids in different regions of the world. The OXA-370 is a plasmid-encoded OXA-_48-like enzyme reported in two distinct regions of Brazil. Recently, we demonstrate that the bla_OXA-370 gene is disseminated among several Enterobacteriaceae species and clones, indicating a high potential for dissemination. In this work, we described for the first time the complete nucleotide sequence of six plasmids harboring the bla_OXA-370 gene. Complete DNA sequencing using the Illumina platform and annotation of the plasmids showed that they belonged to incompatibility groups IncX and had in average 70 kbp. The bla_OXA-370 gene is located in a composite transposon containing four genes encoding transposases, named Tn6435. In this study, highly similar plasmids were detected in different Enterobacteriaceae genera.     

Intrapatient emergence of OXA-247: a novel carbapenemase found in a patient previously infected with OXA-163-producing Klebsiella pneumoniae

Two genetically related Klebsiella pneumoniae strains carrying OXA-type carbapenemases were isolated from a single patient 1 month apart. Kpn163 harboured OXA-163 and Kpn247 a new variant named OXA-247 that showed susceptibility to carbapenems and expanded-spectrum cephalosporins similar to OXA-48. Our epidemiological, biochemical and molecular results suggest the intrapatient emergence of bla_OXA-247 from bla_OXA-163.     

Travel-Related Carbapenemase-Producing Gram-Negative Bacteria in Alberta,Canada: the First 3 Years

We describe here the characteristics of Alberta, Canada, patients with infections or colonizations with carbapenemase-producing Gram-negative bacteria during 2010 to 2013 that were linked to recent travel outside Canada. Antimicrobial susceptibility was determined by broth microdilution, and isolates were characterized using PCR, sequencing, and multilocus sequencing typing. A broth mating study was used to assess the transferability of resistance plasmids, which were subsequently characterized. All the patients (n = 12) included in our study had contact with a health care system while abroad. Most of the patients presented with urinary tract infections (UTIs) and were admitted to hospitals within weeks after their return to Alberta. Secondary spread occurred in 1 case, resulting in the death of another patient. The carbapenemase-producing bacteria (n = 17) consisted of Escherichia coli (sequence type 101 [ST101], ST365, ST405, and ST410) with NDM-1, Klebsiella pneumoniae (ST15, ST16, ST147, ST258, ST340, ST512, and ST972) with NDM-1, OXA-181, KPC-2, and KPC-3, Acinetobacter baumannii with OXA-23, Providencia rettgeri with NDM-1, Enterobacter cloacae with KPC-2, and Citrobacter freundii with NDM-1. The bla_NDM-1 gene was associated with various narrow- (i.e., IncF) and broad- (i.e., IncA/C and IncL/M) host-range plasmids with different addiction factors. Our results show that NDM-producing K. pneumoniae, belonging to a variety of sequence types with different plasmid scaffolds, are regularly imported from India into Alberta. Clinical microbiology laboratories should remain vigilant in detecting bacteria with carbapenemases.     

Outbreak caused by a multi-resistant Klebsiella pneumoniae strain of new sequence type ST341 carrying new genetic environments of aac(6′)-Ib-cr and qnrS1 genes in a neonatal intensive care unit in Spain

An outbreak due to a Klebsiella pneumoniae clone occurred in a neonatal intensive care unit of a Spanish Hospital in which three newborns were infected (all with gestational age ≤29 weeks; two of them died) and seven were colonized (gestational age >32 weeks; none died). One K. pneumoniae strain per patient was further characterized. The 10 strains showed an indistinguishable pulsed-field-gel-electrophoresis pattern, were typed in the phylogenetic group KpI and were ascribed into a new sequence type registered as ST341. All 10 strains presented the same multiple-antibiotic-resistant phenotype, showed extended-spectrum-beta-lactamase production, and harbored the bla_CTX-M-15, bla_SHV-11, bla_OXA-1,aac(6′)-Ib-cr, qnrS1, aac(3)-II, aph(3′)-Ia and aadA5 resistance genes. No class 1 or class 2 integrons were detected. The bla_CTX-M-15 gene presented the following genetic environment: ISEcp1-bla_CTX-M-15-orf477. These strains contained two copies of the aac(6′)-Ib-cr gene included in the following new genetic environments: aac(3)-II-IS26-aac(6′)-Ib-cr-bla_OXA-1 and aac(3)-II-IS26-ΔcatB3-bla_OXA-1-aac(6′)-Ib-cr (registered at GenBank with accession numbers GQ438247 and GQ438248, respectively). The genetic environment of the qnrS1 gene (IS26-ΔISEcl2-qnrS1) (GenBank accession number GQ438249) was also not described previously. The aac(6′)-Ib-cr, qnrS1, bla_CTX-M-15, aac(3)-II, and bla_OXA-1 genes, located in a plasmid of 33.5 kb, could be transferred to Escherichia coli by transformation.     

Investigation of the presence of carbapenemases in carbapenem-resistant Klebsiella pneumoniae strains by MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) and comparison with real-time PCR method

PurposeWe aimed to develop a new procedure for rapid detection of the carbapenemase activity using MALDI-TOF MS, and to determine the sensitivity and specificity of the method. Also, we aimed to determine the distribution of carbapenemase genes among the K.pneumoniae strains isolated in our hospital using real-time PCR.MethodBetween January 2017–February 2019; K. pneumoniae strains(n = 74) isolated from blood culture samples were included. Klebsiella pneumoniae NCTC 13438 was used as a positive control and Escherichia coli ATCC 25922 as a negative control. First, Imipenem, meropenem, and ertapenem MIC values of strains were determined. Then bla_KPC, bla_OXA-48, and/or bla_NDM genes were investigated with PCR. Carbapenemase activity was investigated in strains with the newly developed method using MALDI-TOF MS. The performance of the new method was evaluated for both the second and fourth hours of the incubation period.ResultsWhile 65 strains were found resistant to tested carbapenems, nine of them were susceptible. Of the 65 resistant strains, 57 had bla_OXA-48, 15 had bla_NDM, and four had bla_KPC genes. Bla_OXA-48 and bla_NDM genes were detected together in 11 strains. Bla_OXA-48, bla_NDM, and bla_KPC genes were not detected in any of the susceptible strains. The sensitivity and specificity of MALDI-TOF MS at the second hour were 83.1% and 100%, respectively. At the fourth hour, the sensitivity and specificity of MALDI-TOF MS were 100%. No false-positive results were observed.ConclusionThe sensitivity of the method at the fourth hour was better than the second hour. The false-negative results observed in the second hour disappeared when the incubation period was extended to 4 h. MALDI-TOF MS which is still under development is a fast, cost-effective, promising method for the detection of carbapenemase activity.     

OXA- and GES-type β-lactamases predominate in extensively drug-resistant Acinetobacter baumannii isolates from a Turkish University Hospital

We determined the antibiotic susceptibility and genetic mechanisms of resistance in clinical strains of Acinetobacter baumannii from Istanbul, Turkey. A total of 101 clinical strains were collected between November 2011 and July 2012. Antimicrobial susceptibility was performed using the Vitek 2 Compact system and E-test. Multiplex PCR was used for detecting bla_OXA-51-like, bla_OXA.-_23-like, bla_OXA-40-like and bla_OXA-58-like genes. ISAba1, bla_IMP-like, bla_VIM-like, bla_GES, bla_VEB, bla_PER-2, aac-3-Ia and aac-6'-Ib and NDM-1 genes were detected by PCR and sequencing. By multiplex PCR, all strains were positive for bla_OXA-51, 79 strains carried bla_OXA-23 and one strain carried bla_OXA-40. bla_OXA-51 and bla_OXA-23 were found together in 79 strains. ISAba1 element was detected in 81 strains, and in all cases it was found upstream of bla_OXA-51. GES-type carbapenemases were found in 24 strains (GES-11 in 16 strains and GES-22 in 8 strains) while bla_PER-2, bla_VEB-1, bla_NDM-1, bla_IMP- and bla_VIM-type carbapenemases were not observed. Aminoglycoside modifying enzyme (aac-3-Ia and aac-6'Ib) genes were detected in 13 and 15 strains, respectively. Ninety-seven (96%) A. baumannii strains were defined as MDR and of these, 98% were extensively drug resistant (sensitive only to colistin). Colistin remains the only active compound against all clinical strains. As seen in other regions, OXA-type carbapenemases, with or without an upstream ISAba1, predominate but GES-type carbapenemases also appear to have a significant presence. REP-PCR analysis was performed for molecular typing and all strains were collected into 12 different groups. To our knowledge, this is the first report of GES-11 and OXA-40 in A. baumannii from Turkey.     

Outbreak of KPC-3-producing,and colistin-resistant,Klebsiella pneumoniae infections in two Sicilian hospitals

We report the first outbreak caused by colistin-resistant Klebsiella pneumoniae producing KPC-3 carbapenamase in two Italian hospitals. This spread occurred in 1 month, and was caused by eight colistin-resistant and carbapenem-resistant Klebsiella pneumoniae isolates from eight patients. A further three isolates were obtained from the intestinal tract and pharyngeal colonization. All isolates were multidrug-resistant (MDR), including being resistant to colistin, but they were susceptible to gentamicin and tigecycline. PCR detection showed that all isolates harboured the bla_KPC-3 gene associated with bla_SHV-11, bla_TEM-1 and bla_OXA-9. All K. pneumoniae isolates, genotyped by pulsed-field gel electrophoresis and multilocus sequence typing, belonged to the same sequence type (ST)258 clone. From our data and a review of the international literature, K. pneumoniae ST258 seems to be the most widespread genetic background for KPC dissemination in Europe.     

Four Carbapenem-Resistant Gram-Negative Species Carrying Distinct Carbapenemases in a Single Patient

Carbapenem-resistant Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, and Acinetobacter baumannii were isolated from a single patient, each producing different carbapenemases (NDM-1, KPC-2, IMP, and OXA-23, respectively). The NDM-1-producing E. coli strain was preceded by a clonally related carbapenem-susceptible strain a month earlier, suggesting in vivo acquisition of bla_NDM-1.     

Screening Rectal Swabs for Carbapenemase Genes

In an outbreak setting, we screened 16,296 samples from 3,644 patients by PCR for the presence of bla_OXA-48, bla_VIM, bla_IMP, bla_NDM, and bla_KPC. The bla_OXA-48 gene was found in samples from 43 patients infected with 9 different species of Enterobacteriaceae. Five patients had Pseudomonas aeruginosa isolates containing bla_VIM. The negative predictive value of screening was 100%, and the positive predictive value was 86%.     

Multidrug resistance mediated by co-carriage of extended-spectrum beta-lactamases,AmpC and New Delhi metallo-beta-lactamase-1 genes among carbapenem-resistant Enterobacteriaceae at five Indian medical centres

In this study, we evaluated the coexistence of extended-spectrum beta-lactamases (ESBL), AmpC and New Delhi metallo-beta-lactamase-1 (NDM-1) genes among carbapenem-resistant Enterobacteriaceae (CRE) recovered prospectively from patients at multiple sites. The study included 285 CRE strains from 2782 Gram-negative Bacilli collected from multiple centres during 2007–2010, of which 87 were characterised. Standard and reference laboratory methods were used for resistance determination. Detection of bla_NDM-1, bla_AmpC, bla_TEM, bla_SHV and bla_CTX-M was done by polymerase chain reaction. High levels of antimicrobial resistance observed among study isolates. Co-carriage of ESBLs, AmpC and NDM-1 was 26.3%. Nosocomial origin among the co-carriage isolates was 64.3%, with 9.2% associated mortality.