Acquisition of Broad-Spectrum Cephalosporin Resistance Leading to Colistin Resistance in Klebsiella pneumoniae

An extended-spectrum β-lactamase (ESBL)-producing and colistin-resistant Klebsiella pneumoniae clinical isolate was recovered from a patient who was treated with cefotaxime. This isolate harbored a bla_CTX-M-15 ESBL gene that was associated with an ISEcp1 insertion sequence. Transposition of that tandem occurred within the chromosomal mgrB gene, leading to inactivation of the mgrB gene and consequently to acquired resistance to colistin. We showed here a coselection of colistin resistance as a result of a broad-spectrum cephalosporin selective pressure.     

Loss of Hypermucoviscosity and Increased Fitness Cost in Colistin-Resistant Klebsiella pneumoniae Sequence Type 23 Strains

In this study, we investigated the effects of colistin resistance on virulence and fitness in hypermucoviscous (HV) Klebsiella pneumoniae sequence type 23 (ST23) strains. Colistin-resistant mutants were developed from three colistin-susceptible HV K. pneumoniae ST23 strains. The lipid A structures of strains were analyzed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. Changes in HV were investigated using the string test, and extracellular polysaccharide production was quantified. The expression levels of the phoQ, pmrD, pmrB, pbgP, magA, and p-rmpA2 genes, serum resistance, and biofilm-forming activity were determined. The fitness of colistin-resistant mutants compared to that of the parental strains was examined by determining the competitive index (CI). The colistin-resistant mutants exhibited reduced HV, which was accompanied by decreased formation of capsular polysaccharides (CPS) and reduced expression of genes (magA and p-rmpA2). While there was enhanced expression of pmrD and pbgP in all colistin-resistant derivatives, there were differences in the expression levels of phoQ and pmrB between strains. MALDI-TOF analysis detected the addition of aminoarabinose or palmitate to the lipid A moiety of lipopolysaccharide in the colistin-resistant derivatives. In addition, survival rates in the presence of normal human serum were decreased in the mutant strains, and CI values (0.01 to 0.19) indicated significant fitness defects in the colistin-resistant derivatives compared to the respective parental strains. In hypervirulent HV K. pneumoniae strains, the acquisition of colistin resistance was accompanied by reduced CPS production, impaired virulence, and a significant fitness cost.     

Development and validation of a multiplex PCR assay for identification of the epidemic ST-258/512 KPC-producing Klebsiella pneumoniae clone

The Klebsiella pneumoniae carbapenemase–producing Klebsiella pneumoniae (KPC-KP) sequence type (ST)-258/512 clone is the dominant clone by which KPC has disseminated worldwide. Standard typing methods are time-consuming and are therefore impractical for identification of this clone in the course of an outbreak. Through comparative genomic study, we have previously identified several presumably unique genes of this clone: 1) PILV-like protein (pilv-l), 2) transposase, IS66-family (is-66), and a 3) phage-related protein (prp). Our aims were to 1) test for the presence of these genes using a multiplex PCR in a large, multinational collection of KPC-KP isolates and to 2) validate this assay as a typing method for the identification of the ST-258/512 clone. KPC-KP isolates (n = 160) that included both ST-258/512 (group A, n = 114) and non–ST-258 (group B, n = 46) strains were collected from the following countries: Greece, 20; Israel, 93; Italy, 19; USA, 25; and Colombia, 3. Group B included 30 different STs from various lineages. The pilv-l gene was present in 111/114 of ST-258 isolates, including all of the KPC-negative isolates resulting in a sensitivity of 97%. Using primers for a unique ST-258 pilv-l allele resulted in a specificity of 100%. The sensitivity values of is-66 and prp genes for detecting KPC-KP ST-258 were 83 and 89%, respectively, and the specificity values were 67 and 93%, respectively. PCR for the unique pilv-l ST-258 allele provides a reliable tool for rapid detection of the ST-258 clone. This method can be helpful both in the setting of an outbreak and in a large-scale survey of KPC-KP strains.     

Emergence and Distribution of Plasmids Bearing the blaOXA-51-Like Gene with an Upstream ISAba1 in Carbapenem-Resistant Acinetobacter baumannii Isolates in Taiwan

The bla_OXA-51-like gene with an upstream ISAba1 (ISAba1-bla_OXA-51-like gene) was originally found on the chromosomes of carbapenem-resistant or -susceptible Acinetobacter baumannii isolates. However, a plasmid-borne ISAba1-bla_OXA-51-like gene has recently been identified in Acinetobacter genomic species 13TU and several A. baumannii isolates in Taiwan, and all of the isolates are carbapenem resistant. This study aimed to characterize the plasmids bearing the ISAba1-bla_OXA-51-like gene and their significance in A. baumannii. Among the 117 ISAba1-bla_OXA-51-like-harboring isolates collected from 10 hospitals in Taiwan, 58 isolates (49.6%) from 24 clones had the genes located on plasmids that likely originated from a common progenitor. Among the 58 isolates, four had additional copy of the ISAba1-bla_OXA-51-like gene on their chromosomes. Based on the analysis of these four isolates, the plasmid-located ISAba1-bla_OXA-51-like gene appeared to be acquired via one-ended transposition (Tn6080). The isolates with a plasmid bearing the ISAba1-bla_OXA-51-like gene had higher rates of resistance to imipenem (98% versus 46.6%; P < 0.001) and meropenem (98% versus 69%; P = 0.019) than those with the genes chromosomally encoded, which is most likely due to increased gene dosage provided by the higher copy number of associated plasmids. Transformation with a recombinant plasmid harboring only the ISAba1-bla_OXA-51-like gene was enough to confer a high level of carbapenem resistance to A. baumannii, eliminating the possible contribution of other factors on the original plasmids. This study demonstrated that the carbapenem resistance-associated plasmids carrying the ISAba1-bla_OXA-51-like gene are widespread in A. baumannii strains in Taiwan.Acinetobacter baumannii has emerged as a major pathogen of nosocomial infections in immunocompromised patients and is associated with a high mortality rate (7). The management of this pathogen has become a significant challenge due to the increased emergence of carbapenem-resistant strains (19, 22). It has been shown that most of the carbapenem resistance in A. baumannii is due to the production of carbapenemases, especially those belonging to carbapenem-hydrolyzing class D beta-lactamases (CHDLs), which are encoded by the bla_OXA-51-like, bla_OXA-23-like, bla_OXA-24-like, and bla_OXA-58-like genes (19). Among these CHDL genes, the bla_OXA-51-like gene is intrinsic to and was originally located on the chromosome of A. baumannii (7, 18, 24).The CHDLs have only a weak carbapenem-hydrolyzing activity (1, 19). However, overexpression of these CHDL genes, driven mostly by promoters provided by their upstream insertion sequences (ISs), is one of the means by which A. baumannii acquires a high level of carbapenem resistance. For example, ISAba1 upstream of the bla_OXA-51-like gene is associated with the overexpression of the bla_OXA-51-like gene and carbapenem resistance in A. baumannii (12, 23). However, some isolates harboring the bla_OXA-51-like gene with an upstream ISAba1 (ISAba1-bla_OXA-51-like gene) are still susceptible to carbapenem (9, 14, 16). The reason for this discrepancy in carbapenem susceptibility levels in isolates harboring the ISAba1-bla_OXA-51-like gene has not been completely clarified. In some isolates, the high level of resistance might be due to interplay between the overexpression of the bla_OXA-51-like gene and other mechanisms, such as overexpression of an efflux pump (12, 19). However, the overexpression of the CHDL genes by themselves can occasionally confer a high level of carbapenem resistance, especially when the resistance determinants are located on a plasmid, and this is likely due to the higher gene dosage provided by the higher copy number associated with plasmids (3). Recently, we have identified several A. baumannii isolates in Taiwan that harbor plasmid-borne ISAba1-bla_OXA-51-like genes (ISAba1-bla_OXA-82) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GQ352402","term_id":"255076961","term_text":"GQ352402"}}GQ352402). Moreover, a plasmid bearing the ISAba1-bla_OXA-51-like gene (ISAba1-bla_OXA-138) has also been indentified in an Acinetobacter genomic species 13TU isolate (15). The mobilization of the ISAba1-bla_OXA-51-like gene to plasmids and dissemination of these plasmids will further complicate the management of A. baumannii infections. This study aimed to characterize the spread of these plasmids and their significance in A. baumannii isolates in Taiwan.     

Emergence and dissemination of colistin-resistant Klebsiella pneumoniae isolates expressing OXA-48 plus CTX-M-15 in patients not previously treated with colistin in a Spanish university hospital

Dissemination of multidrug-resistant Klebsiella pneumoniae in the hospital environment represents a primary target of resistance containment and stewardship programs. At present, polymyxins, mostly in combination, exemplify a last-resort alternative. Colistin-resistant K. pneumoniae isolates harboring OXA-48 plus CTX-M-15 (n?=?21) with the simultaneous colistin-susceptible counterparts (n?=?9) were recovered from 14 hospitalized patients (January 2014–January 2015) admitted in different wards. In most cases, patients had not previously received colistin. Genetic relatedness experiments demonstrated that 93% (28/30) of isolates belonged to the ST11 high-risk clone. Heteroresistance and the fitness cost of colistin resistance were addressed in susceptible and resistant isolates as well as in in vitro–obtained stable mutants, and results appeared to be strain dependent. Whole genome sequencing demonstrated molecular changes in pmrA, pmrB, and mgrB genes. Plasmid-mediated colistin resistance genes were not found. Colistin resistance in multidrug-resistant K. pneumoniae isolates should be continuously monitored to detect its potential emergence, even in patients not previously exposed to colistin.     

Polymyxin Resistance Caused by mgrB Inactivation Is Not Associated with Significant Biological Cost in Klebsiella pneumoniae

The inactivation of the mgrB gene, which encodes a negative-feedback regulator of the PhoPQ signaling system, was recently shown to be a common mutational mechanism responsible for acquired polymyxin resistance among carbapenemase-producing Klebsiella pneumoniae strains from clinical sources. In this work, we show that mgrB mutants can easily be selected in vitro from different K. pneumoniae lineages, and mgrB inactivation is not associated with a significant biological cost.     

Detection of plasmid-mediated colistin resistance,mcr-1 gene,in Escherichia coli isolated from high-risk patients with acute leukemia in Spain

BackgroundBacterial infections in immunocompromised patients are associated with a high mortality and morbidity rate. In this high-risk group, the presence of multidrug-resistant (MDR) bacteria, particularly bacteria that harbor a transferable antibiotic resistance gene, complicates the management of bacterial infections. In this study, we investigated the presence of the transferable colistin resistance mcr genes in patients with leukemia in Spain.Methods217 fecal samples collected in 2013–2015 from 56 patients with acute leukemia and colonized with MDR Enterobacteriaceae strains, were screened on September 2017 for the presence of the colistin resistance mcr genes (mcr-1 to -5) by multiplex PCR. mcr positive strains selected on LBJMR and MacConkey supplemented with colistin (2 μg/ml) media were phenotypically and molecularly characterized by antimicrobial susceptibility testing, minimum inhibitory concentration, multilocus sequence typing and plasmid characterization.ResultsAmong 217 fecal samples, 5 samples collected from 3 patients were positive for the presence of the mcr-1 colistin-resistance gene. Four Escherichia coli strains were isolated and exhibited resistance to colistin with MIC = 4 μg/ml. Other genes conferring the resistance to β-lactam antibiotics have also been identified in mcr-1 positive strains, including bla_TEM-206 and bla_TEM-98. Three different sequence types were identified, including ST1196, ST140 and ST10. Plasmid characterization allowed us to detect the mcr-1 colistin resistance gene on conjugative IncP plasmid type.ConclusionTo the best of our knowledge, we have identified the mcr-1 gene for the first time in leukemia patients in Spain. In light of these results, strict measures have been implemented to prevent its dissemination.     

Mutations of the ompK36 Porin Gene and Promoter Impact Responses of Sequence Type 258, KPC-2-Producing Klebsiella pneumoniae Strains to Doripenem and Doripenem-Colistin

Doripenem-colistin exerts synergy against some, but not all, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains in vitro. We determined if doripenem MICs and/or ompK36 porin gene mutations impacted the responses of 23 sequence type 258 (ST258), KPC-2-producing strains to the combination of doripenem (8 μg/ml) and colistin (2 μg/ml) during time-kill assays. The median doripenem and colistin MICs were 32 and 4 μg/ml. Doripenem MICs did not correlate with KPC-2 expression levels. Five and 18 strains had wild-type and mutant ompK36, respectively. The most common mutations were IS5 promoter insertions (n = 7) and insertions encoding glycine and aspartic acid at amino acid (aa) positions 134 and 135 (ins aa134-135 GD; n = 8), which were associated with higher doripenem MICs than other mutations or wild-type ompK36 (all P values ≤ 0.04). Bactericidal activity (24 h) was achieved by doripenem-colistin against 12%, 43%, and 75% of ins aa134-135 GD, IS5, and wild-type/other mutants, respectively (P = 0.04). Doripenem-colistin was more active in time-kill studies than colistin at 12 and 24 h if the doripenem MIC was ≤8 μg/ml (P = 0.0007 and 0.09, respectively), but not if the MIC was >8 μg/ml (P = 0.10 and 0.16). Likewise, doripenem-colistin was more active at 12 and 24 h against the wild type/other mutants than ins aa134-135 GD or IS5 mutants (P = 0.007 and 0.0007). By multivariate analysis, the absence of ins aa134-135 GD or IS5 mutations was the only independent predictor of doripenem-colistin responses at 24 h (P = 0.002). In conclusion, ompK36 genotypes identified ST258 KPC-K. pneumoniae strains that were most likely to respond to doripenem-colistin.     

Resistance to Colistin Associated with a Single Amino Acid Change in Protein PmrB among Klebsiella pneumoniae Isolates of Worldwide Origin

A series of colistin-resistant Klebsiella pneumoniae isolates recovered from different countries was investigated in order to evaluate the involvement of the PmrA/PmrB two-component system in this resistance. Six isolates possessed a mutated PmrB protein, which is encoded by the pmrB gene, part of the pmrCAB operon involved in lipopolysaccharide modification. The same amino acid substitution (Thr157Pro) in PmrB was identified in the six isolates. The six isolates belonged to four distinct clonal groups, recovered in South Africa (sequence type 14 [ST14]), Turkey (ST101), and Colombia (ST258 and ST15). Three out of the four clones produced a carbapenemase, OXA-181, OXA-48, or KPC-3, while a single isolate did not produce any carbapenemase. Expression assays revealed an overexpression of the pmrA (70-fold), pmrB (70-fold), pmrC (170-fold), and pmrK (40-fold) genes in the pmrB-mutated isolate compared to expression of the pmrB wild-type isogenic K. pneumoniae isolate, confirming that the PmrB substitution was responsible for increased expression levels of those genes. Complementation assays leading to the expression of a wild-type PmrB protein restored the susceptibility to colistin in all isolates, confirming that the substitution in PmrB was responsible for the resistance phenotype. This study identified a key amino acid located in the PmrB protein as being responsible for the overexpression of pmrCAB and pmrHFIJKLM operons, leading to resistance to colistin.     

ISEcp1-mediated transposition of qnrB-like gene in Escherichia coli

A novel QnrB-like plasmid-mediated resistance determinant, QnrB19, was identified from an Escherichia coli clinical isolate from Colombia. Its gene was associated with an ISEcp1-like insertion element that did not act as a promoter for its expression. Using an in vitro model of transposition, we showed that the ISEcp1-like element was able to mobilize the qnrB19 gene.     

Increasing prevalence of blaOXA-23-carrying Acinetobacter baumannii and the emergence of blaOXA-182-carrying Acinetobacter nosocomialis in Korea

Carbapenem-resistant Acinetobacter spp. have been increasingly reported worldwide with the production of OXA-type carbapenemases as the main mechanism of carbapenem resistance. The prevalent bla_OXA genes are known to vary significantly depending on time and place of isolation. We investigated the prevalence of bla_OXA genes by PCR in Acinetobacter spp. isolated in Korea. Among a total of 336 isolates collected from Hospital A from 2002 to 2011, the overall proportion of bla_OXA-23-like, ISAba1-associated bla_OXA-51-like, and bla_OXA-182 genes were 44.0%, 49.7%, and 5.1%, respectively. The bla_OXA-58-like gene was detected in only 1 isolate. A drastic increase in Acinetobacter isolates with bla_OXA-23-like genes and a decrease in isolates harboring ISAba1-associated bla_OXA-51-like genes have been observed since the mid-2000s. The bla_OXA-23-like genes were detected in all carbapenem-nonsusceptible isolates collected in 2011 from 9 hospitals. The OXA-182, which belongs to the fifth group of OXA-type carbapenemase, was detected in Acinetobacter baumannii isolates recovered as early as 2002. It is worrisome results that bla_OXA-182-carrying Acinetobacter nosocomialis has emerged and caused outbreaks of infection.     

Genetic structures at the origin of acquisition and expression of the carbapenem-hydrolyzing oxacillinase gene blaOXA-58 in Acinetobacter baumannii

Genetic structures surrounding the carbapenem-hydrolyzing oxacillinase gene bla_OXA-58 were characterized in a series of OXA-58-positive Acinetobacter baumannii strains isolated from different countries. We showed that in most of the cases, acquisitions of the bla_OXA-58-containing overall structure, including insertion sequence elements, may be likely the results of recombination events. In type strain A. baumannii MAD, the genetic structure surrounding the bla_OXA-58 gene was bracketed by two 27-bp repeated sequences. The isolation of a clonally related OXA-58-negative A. baumannii isolate that possessed the same plasmid backbone as A. baumannii MAD but lacked this genetic structure indicated that the mechanism of acquisition could be reversible. Parts of the structure identified in A. baumannii MAD were conserved in other bla_OXA-58-positive isolates from various European countries. Primer extension experiments showed that bla_OXA-58 expression was related to promoter sequences brought by different insertion sequence elements, such as an ISAba3-like element, ISAba1, ISAba2, and IS18. This work identified novel structures at the origin of acquisition and expression of a carbapenem-hydrolyzing β-lactamase identified in non-clonally related A. baumannii isolates.     

Evolution of the ISCR3 group of ISCR elements

The ISCR elements ISCR3, ISCR4, ISCR5, ISCR14, and ISCR16 all share a percent G+C of 68 to 69%. They also share between 75% and 97% identity in their transposase open reading frames. Furthermore, with the exception of ISCR5, they are all found adjacent to sections of groEL that display the highest identity to the same gene from Xanthomonas spp. The combined information is consistent with the descent from an ancestral ISCR element in a Xanthomonas-like organism.     

Genetic basis of multidrug resistance in Acinetobacter baumannii clinical isolates at a tertiary medical center in Pennsylvania

A total of 49 unique clinical isolates of multidrug-resistant (MDR) Acinetobacter baumannii identified at a tertiary medical center in Pittsburgh, Pennsylvania, between August 2006 and September 2007 were studied for the genetic basis of their MDR phenotype. Approximately half of all A. baumannii clinical isolates identified during this period qualified as MDR, defined by nonsusceptibility to three or more of the antimicrobials routinely tested in the clinical microbiology laboratory. Among the MDR isolates, 18.4% were resistant to imipenem. The frequencies of resistance to amikacin and ciprofloxacin were high at 36.7% and 95.9%, respectively. None of the isolates was resistant to colistin or tigecycline. The presence of the carbapenemase gene bla_OXA-23 and the 16S rRNA methylase gene armA predicted high-level resistance to imipenem and amikacin, respectively. bla_OXA-23 was preceded by insertion sequence ISAba1, which likely provided a potent promoter activity for the expression of the carbapenemase gene. The structure of the transposon defined by ISAba1 differed from those reported in Europe, suggesting that ISAba1-mediated acquisition of bla_OXA-23 may occur as an independent event. Typical substitutions in the quinolone resistance-determining regions of the gyrA and parC genes were observed in the ciprofloxacin-resistant isolates. Plasmid-mediated quinolone resistance genes, including the qnr genes, were not identified. Fifty-nine percent of the MDR isolates belonged to a single clonal group over the course of the study period, as demonstrated by pulsed-field gel electrophoresis.     

Molecular Characterization of blaNDM-1 in a Sequence Type 235 Pseudomonas aeruginosa Isolate from France

An NDM-1 carbapenemase-producing Pseudomonas aeruginosa isolate was recovered from a patient hospitalized in France after a previous hospitalization in Serbia. Genetic studies revealed that the bla_NDM-1 gene was surrounded by insertion sequence ISAba125 and a truncated bleomycin resistance gene. This bla_NDM-1 region was a part of the variable region of a new complex class 1 integron bearing IS common region 1 (ISCR1). The presence of ISPa7 upstream of this integron suggests insertion in a chromosomally located Tn402-like structure.     

First Report of KPC-2 Carbapenemase-Producing Klebsiella pneumoniae in Japan

We investigated a novel Japanese isolate of sequence type 11 (ST11), the Klebsiella pneumoniae carbapenemase-2 (KPC-2)-producing K. pneumoniae strain Kp3018, which was previously obtained from a patient treated at a Brazilian hospital. This strain was resistant to various antibiotic classes, including carbapenems, and harbored the gene bla_KPC-2, which was present on the transferable plasmid of ca. 190 kb, in addition to the bla_CTX-M-15 gene. Furthermore, the ca. 2.3-kb sequences (ISKpn8-bla_KPC-2–ISKpn6-like), encompassing bla_KPC-2, were found to be similar to those of K. pneumoniae strains from China.