Global spread characteristics of CTX-M-type extended-spectrum ß-lactamases: A genomic epidemiology analysis.

BACKGROUND: Extended-spectrum ß-lactamases (ESBLs) producing bacteria have spread worldwide and become a global public health concern. Plasmid-mediated transfer of ESBLs is an important route for resistance acquisition. METHODS: We collected 1345 complete sequences of plasmids containing CTX-Ms from public database. The global transmission pattern of plasmids and evolutionary dynamics of CTX-Ms have been inferred. We applied the pan-genome clustering based on plasmid genomes and evolution analysis to demonstrate the transmission events. FINDINGS: Totally, 48 CTX-Ms genotypes and 186 incompatible types of plasmids were identified. The geographical distribution of CTX-Ms showed significant differences across countries and continents. CTX-M-14 and CTX-M-55 were found to be the dominant genotypes in Asia, while CTX-M-1 played a leading role in Europe. The plasmids can be divided into 12 lineages, some of which forming distinct geographical clusters in Asia and Europe, while others forming hybrid populations. The Inc types of plasmids are lineage-specific, with the CTX-M-1_IncI1-I (Alpha) and CTX-M-65_IncFII (pHN7A8)/R being the dominant patterns of cross-host and cross-regional transmission. The IncI-I (Alpha) plasmids with the highest number, were presumed to form communication groups in Europe-Asia and Asia-America-Oceania, showing the transmission model as global dissemination and regional microevolution. Meanwhile, the main kinetic elements of blaCTX-Ms showed genotypic preferences. ISEcpl and IS26 were most frequently involved in the transfer of CTX-M-14 and CTX-M-65, respectively. IS15 has become a crucial participant in mediating the dissemination of blaCTX-Ms. Interestingly, blaTEM and blaCTX-Ms often coexisted in the same transposable unit. Furthermore, antibiotic resistance genes associated with aminoglycosides, sulfonamides and cephalosporins showed a relatively high frequency of synergistic effects with CTX-Ms. CONCLUSIONS: We recognized the dominant blaCTX-Ms and mainstream plasmids of different continents. The results of this study provide support for a more effective response to the risks associated with the evolution of blaCTX-Ms-bearing plasmids, and lay the foundation for genotype-specific epidemiological surveillance of resistance, which are of important public health implications.

An Intra-Hospital Spread of Colistin-Resistant K. pneumoniae Isolates-Epidemiological, Clinical, and Genetic Analysis.

Background and Objective: Klebsiella pneumoniae appears to be a significant problem due to its ability to accumulate antibiotic-resistance genes. After 2013, alarming colistin resistance rates among carbapenem-resistant K. pneumoniae have been reported in the Balkans. The study aims to perform an epidemiological, clinical, and genetic analysis of a local outbreak of COLr CR-Kp. Material and Methods: All carbapenem-resistant and colistin-resistant K. pneumoniae isolates observed among patients in the ICU unit of Military Medical Academy, Sofia, from 1 January to 31 October 2023, were included. The results were analyzed according to the EUCAST criteria. All isolates were screened for blaVIM, blaIMP, blaKPC, blaNDM, and blaOXA-48. Genetic similarity was determined using the Dice coefficient as a similarity measure and the unweighted pair group method with arithmetic mean (UPGMA). mgrB genes and plasmid-mediated colistin resistance determinants (mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5) were investigated. Results: There was a total of 379 multidrug-resistant K. pneumoniae isolates, 88% of which were carbapenem-resistant. Of these, there were nine (2.7%) colistin-resistant isolates in six patients. A time and space cluster for five patients was found. Epidemiology typing showed that two isolates belonged to clone A (pts. 1, 5) and the rest to clone B (pts. 2-4) with 69% similarity. Clone A isolates were coproducers of blaNDM-like and blaOXA-48-like and had mgrB-mediated colistin resistance (40%). Clone B isolates had only blaOXA-48-like and intact mgrB genes. All isolates were negative for mcr-1, -2, -3, -4, and -5 genes. Conclusions: The study describes a within-hospital spread of two clones of COLr CR-Kp with a 60% mortality rate. Clone A isolates were coproducers of NDM-like and OXA-48-like enzymes and had mgrB-mediated colistin resistance. Clone B isolates had only OXA-48-like enzymes and intact mgrB genes. No plasmid-mediated resistance was found. The extremely high mortality rate and limited treatment options warrant strict measures to prevent outbreaks.

Genomic Insights Into the Mechanism of Carbapenem Resistance Dissemination in Enterobacterales From a Tertiary Public Heath Setting in South Asia.

SUMMARY: 10.6% patients were CRE positive. Only 27% patients were prescribed at least 1 antibiotic to which infecting pathogen was susceptible. Burn and ICU admission and antibiotics exposures facilitate CRE acquisition. Escherichia coli ST167 was the dominant CRE clone. BACKGROUND: Given the high prevalence of multidrug resistance (MDR) across South Asian (SA) hospitals, we documented the epidemiology of carbapenem-resistant Enterobacterales (CRE) infections at Dhaka Medical College Hospital between October 2016 and September 2017. METHODS: We enrolled patients and collected epidemiology and outcome data. All Enterobacterales were characterized phenotypically and by whole-genome sequencing. Risk assessment for the patients with CRE was performed compared with patients with carbapenem-susceptible Enterobacterales (CSE). RESULTS: 10.6% of all 1831 patients with a clinical specimen collected had CRE. In-hospital 30-day mortality was significantly higher with CRE [50/180 (27.8%)] than CSE [42/312 (13.5%)] (P = .001); however, for bloodstream infections, this was nonsignificant. Of 643 Enterobacterales isolated, 210 were CRE; blaNDM was present in 180 isolates, blaOXA-232 in 26, blaOXA-181 in 24, and blaKPC-2 in 5. Despite this, ceftriaxone was the most commonly prescribed empirical antibiotic and only 27% of patients were prescribed at least 1 antibiotic to which their infecting pathogen was susceptible. Significant risk factors for CRE isolation included burns unit and intensive care unit admission, and prior exposure to levofloxacin, amikacin, clindamycin, and meropenem. Escherichia coli ST167 was the dominant CRE clone. Clustering suggested clonal transmission of Klebsiella pneumoniae ST15 and the MDR hypervirulent clone, ST23. The major trajectories involved in horizontal gene transfer were IncFII and IncX3, IS26, and Tn3. CONCLUSIONS: This is the largest study from an SA public hospital combining outcome, microbiology, and genomics. The findings indicate the urgent implementation of targeted diagnostics, appropriate antibiotic use, and infection-control interventions in SA public institutions.

Plasmid-Mediated Ciprofloxacin Resistance Imparts a Selective Advantage on Escherichia coli ST131.

Escherichia coli ST131 is a recently emerged antibiotic resistant clone responsible for high rates of urinary tract and bloodstream infections. Despite its global dominance, the precise mechanisms that have driven the rapid dissemination of ST131 remain unknown. Here, we show that the plasmid-associated resistance gene encoding the AAC(6')-Ib-cr enzyme that inactivates the fluoroquinolone (FQ) antibiotic ciprofloxacin is present in >70% of strains from the most rapidly expanding subgroup of multidrug resistant ST131. Using a series of genome-edited and plasmid-cured isogenic strains, we demonstrate that the aac(6')-Ib-cr gene confers a selective advantage on ST131 in the presence of ciprofloxacin, even in strains containing chromosomal GyrA and ParC FQ-resistance mutations. Further, we identify a pattern of emerging carbapenem resistance in other common E. coli clones carrying both aac(6')-Ib-cr and chromosomal FQ-resistance mutations, suggesting this dual resistance combination may also impart a selective advantage on these non-ST131 antibiotic resistant lineages.

Multidrug-Resistant Gram-Negative Bacteria in Burn Patients.

Patients with burn injuries are at high risk for infectious complications, and infections are the most common cause of death after the first 72 h of hospitalization. Hospital-acquired infections caused by multidrug resistant (MDR) Gram-negative bacteria (GNB) in this population are concerning. Here, we evaluated carriage with MDR GNB in patients in a large tertiary-care burn intensive care unit. Twenty-nine patients in the burn unit were screened for intestinal carriage. Samples were cultured on selective media. Median time from admission to the burn unit to first sample collection was 9 days (IQR 5 - 17 days). In 21 (72%) patients, MDR GNB were recovered; the most common bacterial species isolated was Pseudomonas aeruginosa, which was found in 11/29 (38%) of patients. Two of these patients later developed bloodstream infections with P. aeruginosa. Transmission of KPC-31-producing ST22 Citrobacter freundii was detected. Samples from two patients grew genetically similar C. freundii isolates that were resistant to ceftazidime-avibactam. On analysis of whole-genome sequencing, blaKPC-31 was part of a Tn4401b transposon that was present on two different plasmids in each C. freundii isolate. Plasmid curing experiments showed that removal of both copies of blaKPC-31 was required to restore susceptibility to ceftazidime-avibactam. In summary, MDR GNB colonization is common in burn patients and patient-to-patient transmission of highly resistant GNB occurs. These results emphasize the ongoing need for infection prevention and antimicrobial stewardship efforts in this highly vulnerable population.

Modified Drug-Susceptibility Testing and Screening Culture Agar for Colistin-Susceptible Enterobacteriaceae Isolates Harboring a Mobilized Colistin Resistance Gene mcr-9.

Three isolates of the Enterobacter cloacae complex harboring mcr-9, a member of the colistin resistance mcr gene family encoded on plasmids, were susceptible to colistin, with MICs of 0.125 to 0.5 µg/mL in standard broth microdilution (BMD) tests using cation-adjusted Mueller-Hinton broth (CA-MHB) in accordance with European Committee on Antimicrobial Susceptibility Testing guidelines. In contrast, their MICs for colistin were significantly higher (4 to 128 µg/mL) when BMD tests were performed using brain-heart infusion (BHI) medium, Luria-Bertani (LB) broth, tryptic soy broth (TSB), or CA-MHB supplemented with casein, tryptonen or peptone. Colistin significantly induced mcr-9 expression in a dose-dependent manner when these mcr-9-positive isolates were cultured in BHI or CA-MHB supplemented with peptone/casein. Pretreatment of mcr-9-positive isolates and Escherichia coli DH5α harboring mcr-9 with colistin significantly increased their survival rates against LL-37, a human antimicrobial peptide. Electrospray ionization time-of-flight mass spectrometry analysis showed that a lipid A moiety of lipopolysaccharide was partially modified by phosphoethanolamine in E. coli DH5α harboring mcr-9 when treated with colistin. Of 93 clinical isolates of Enterobacteriaceae, only the mcr-9-positive isolates showed MICs to colistin that were at least 32 times higher in BHI than in CA-MHB. These mcr-9-positive isolates grew on a modified BHI agar, MCR9-JU, containing 3 µg/mL colistin. These results suggest that the BMD method using BHI is useful when performed together with the BMD method using CA-MHB to detect mcr-9-positive isolates and that MCR9-JU agar is useful in screening for Enterobacteriaceae isolates harboring mcr-9 and other colistin-resistant isolates.

Quantifying the impact of treatment history on plasmid-mediated resistance evolution in human gut microbiota.

To understand how antibiotic use affects the risk of a resistant infection, we present a computational model of the population dynamics of gut microbiota including antibiotic resistance-conferring plasmids. We then describe how this model is parameterized based on published microbiota data. Finally, we investigate how treatment history affects the prevalence of resistance among opportunistic enterobacterial pathogens. We simulate treatment histories and identify which properties of prior antibiotic exposure are most influential in determining the prevalence of resistance. We find that resistance prevalence can be predicted by 3 properties, namely the total days of drug exposure, the duration of the drug-free period after last treatment, and the center of mass of the treatment pattern. Overall this work provides a framework for capturing the role of the microbiome in the selection of antibiotic resistance and highlights the role of treatment history for the prevalence of resistance.

Exploring the Global Spread of Klebsiella grimontii Isolates Possessing blaVIM-1 and mcr-9.

The spread of plasmid-mediated carbapenemases within Klebsiella oxytoca is well-documented. In contrast, data concerning the closely related species Klebsiella grimontii are scarce. In fact, despite the recent report of the first blaKPC-2-producing K. grimontii, nothing is known about its clonality and antibiotic resistance patterns. In a retrospective search in our collection, we identified 2 blaVIM-positive K. oxytoca strains. Whole-genome sequencing with both Illumina and Nanopore indicated that our strains actually belonged to K. grimontii and were of sequence type 172 (ST172) and ST189. Moreover, the two strains were associated with 297-kb IncHI2/HI2A-pST1 and 90.6-kb IncFII(Yp) plasmids carrying blaVIM-1 together with mcr-9 and blaVIM-1, respectively. In the IncHI2/HI2A plasmid, blaVIM-1 was located in a class 1 integron (In110), while mcr-9 was associated with the qseC-qseB-like regulatory elements. Overall, this plasmid was shown to be very similar to those carried by other Enterobacterales isolated from food and animal sources (e.g., Salmonella and Enterobacter spp. detected in Germany and Egypt). The IncFII(Yp) plasmid was unique, and its blaVIM-1 region was associated with a rare integron (In1373). Mapping of In1373 indicated a possible origin in Austria from an Enterobacter hormaechei carrying a highly similar plasmid. Core-genome phylogenies indicated that the ST172 K. grimontii belonged to a clone of identical Swedish and Swiss strains (≤15 single nucleotide variants [SNVs] to each other), whereas the ST189 strain was sporadic. Surveillance of carbapenemase-producing K. oxytoca strains should be reinforced to detect and prevent the dissemination of new species belonging to the Klebsiella genus.

Characterization of Proteobacterial Plasmid Integron-Encoded qac Efflux Pump Sequence Diversity and Quaternary Ammonium Compound Antiseptic Selection in Escherichia coli Grown Planktonically and as Biofilms.

Qac efflux pumps from proteobacterial multidrug-resistant plasmids are integron encoded and confer resistance to quaternary ammonium compound (QAC) antiseptics; however, many are uncharacterized and misannotated. A survey of >2,000 plasmid-carried qac genes identified 37 unique qac sequences that correspond to one of five representative motifs: QacE, QacEΔ1, QacF/L, QacH/I, and QacG. Antimicrobial susceptibility testing of each cloned qac member in Escherichia coli highlighted distinctive antiseptic susceptibility patterns that were most prominent when cells grew as biofilms.

Multidrug-resistant Raoultella ornithinolytica misidentified as Klebsiella oxytoca carrying blaOXA ß-lactamases: antimicrobial profile and genomic characterization.

Class D ß-lactamases OXA-232 and OXA-48 hydrolyze penicillin, cephalosporins and carbapenems, limiting the pharmacological therapeutics in bacteraemia. OXA producer microorganisms are considered a great emergent threat, especially in nosocomial environments. To determine the resistance profile and genomic characterization of two isolates initially identified as potential carbapenemase-producer Klebsiella oxytoca in a third level hospital. Automated platform BD Phoenix-100 System was used to identify and to biochemically characterize both isolates. Furthermore, the resistance profile was determined through CLSI methods and the whole genome sequences were obtained using Next-Generation Sequencing. Resistance genes were analyzed, and the virtual fingerprinting was determined to corroborate the similarity with related bacteria. Both strains correspond to Raoultella ornithinolytica carrying OXA 232 and OXA-48 genes, confirming the class D ß-lactamases assay results. Here, we present the genetic and phenotypic analysis of multidrug resistance R. ornithinolytica, representing the first report in Mexico.

Mobilized colistin resistance (mcr) genes from 1 to 10: a comprehensive review.

At the present time, the polymyxin antibiotic colistin is considered a last-line treatment option for severe human infections caused by multi-drug and carbapenem-resistant Gram-negative bacteria. Lately, the vast spread of colistin resistance among bacteria has got great attention worldwide due to its significant role as the last refuge in treating diseases caused by the resistant infectious agents. Therefore, the discovery of plasmid-mediated mobile colistin resistance (mcr) genes raised global public health concerns as they can spread by horizontal transfer and have chances of global dissemination. To date, ten slightly different variants of the mcr-1 gene (mcr-1 to mcr-10) have been identified in different bacteria isolated from animals, foods, farms, humans, and the environment. Therefore, the issue of mcr spread is growing and worsening day after day. In this backdrop, the current article presents an overview of mcr variants, their spread, and the resistance mechanisms they confer. Hence, this paper will advance our knowledge about colistin resistance while supporting the efforts toward better stewardship and proper usage of antimicrobials.

A Multispecies Cluster of VIM-1 Carbapenemase-Producing Enterobacterales Linked by a Novel, Highly Conjugative, and Broad-Host-Range IncA Plasmid Forebodes the Reemergence of VIM-1.

In this study, we investigated VIM-1-producing Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Citrobacter freundii, and Enterobacter cloacae strains, isolated in 2019 during a period of active surveillance of carbapenem-resistant Enterobacterales in a large university hospital in Italy. VIM-1-producing strains colonized the gut of patients, with up to three different VIM-1-positive bacterial species isolated from a single rectal swab, but also caused bloodstream infection in one colonized patient. In the multispecies cluster, blaVIM-1 was identified in a 5-gene cassette class 1 integron, associated with several genetic determinants, including the blaSHV-12, qnrS1, and mph(A) genes, located on a highly conjugative and broad-host-range IncA plasmid. The characteristics and origin of this IncA plasmid were studied.

Rapid Increase of CTX-M-Producing Shigella sonnei Isolates in Switzerland Due to Spread of Common Plasmids and International Clones.

The Swiss Centre for Antibiotic Resistance (ANRESIS) has recently noted an increase of extended-spectrum cephalosporin-resistant (ESC-R) Shigella sonnei isolates nationwide (3.8% in 2016 versus 37.5% in 2019). To understand this phenomenon, we analyzed 25 representative isolates (of which 14 were ESC-R) collected in Switzerland during 2016 to 2019. Whole-genome sequencing was achieved using both the Illumina and the Nanopore platforms. Both ESC-R and extended-spectrum cephalosporin-susceptible isolates belonged to sequence type 152 (ST152). The ESC-R isolates carried blaCTX-M-3 in IncI1-pST57 (n = 5), blaCTX-M-15 in IncFII (F2:A-:B-) (n = 5), blaCTX-M-15 in IncI1-pST16, and blaCTX-M-27, blaCTX-M-55, or blaCTX-M-134 in other IncFII plasmids (n = 1 each). Plasmids having the same bla and Inc group exhibited high degrees of genetic identity to each other but also to plasmids previously reported in other Enterobacterales Core-genome analysis showed that there were 4 main clusters, each of which included strains that differed by <58 single nucleotide variants (SNVs) and that consisted of both blaCTX-M-positive and blaCTX-M-negative isolates. Moreover, most isolates belonging to the same cluster shared an identical core-genome sequence type (cgST). For instance, cluster 1 included 4 isolates of cgST113036, of which only 3 harbored the IncI1-pST57 blaCTX-M-3-positive plasmid. The 25 S. sonnei isolates were also subjected to phylogenetic comparison with deposited international strains. As a result, matching isolates (isolates that had the same cgST and that differed by <8 SNVs) have been reported in the United Kingdom, the United States, France, and the Netherlands. Overall, our results suggest that some common S. sonnei clusters can spread between continents and can be imported into other nations after international trips. Such clusters include, in part, isolates that do not possess blaESBL-harboring plasmids, indicating their tendency to acquire them from other Enterobacterales.

Identification of a Carbapenemase-Producing Hypervirulent Klebsiella pneumoniae Isolate in the United States.

We report on a carbapenemase-producing hypervirulent Klebsiella pneumoniae (CP-hvKP) isolate collected from a U.S. patient at an outpatient clinic. The isolate was identified as K. pneumoniae serotype K1 sequence type 23 and included both a hypervirulence (with rmpA, rmpA2 iroBCDN, peg-344, and iucABCD-iutA genes) and a carbapenemase-encoding (blaKPC-2) plasmid. The emergence of CP-hvKP underscores the importance of clinical awareness of this pathotype and the need for continued monitoring of CP-hvKP in the United States.

Spreading Patterns of NDM-Producing Enterobacteriaceae in Clinical and Environmental Settings in Yangon, Myanmar.

The spread of carbapenemase-producing Enterobacteriaceae (CPE), contributing to widespread carbapenem resistance, has become a global concern. However, the specific dissemination patterns of carbapenemase genes have not been intensively investigated in developing countries, including Myanmar, where NDM-type carbapenemases are spreading in clinical settings. In the present study, we phenotypically and genetically characterized 91 CPE isolates obtained from clinical (n = 77) and environmental (n = 14) samples in Yangon, Myanmar. We determined the dissemination of plasmids harboring genes encoding NDM-1 and its variants using whole-genome sequencing and plasmid analysis. IncFII plasmids harboring blaNDM-5 and IncX3 plasmids harboring blaNDM-4 or blaNDM-7 were the most prevalent plasmid types identified among the isolates. The IncFII plasmids were predominantly carried by clinical isolates of Escherichia coli, and their clonal expansion was observed within the same ward of a hospital. In contrast, the IncX3 plasmids were found in phylogenetically divergent isolates from clinical and environmental samples classified into nine species, suggesting widespread dissemination of plasmids via horizontal transfer. Half of the environmental isolates were found to possess IncX3 plasmids, and this type of plasmid was confirmed to transfer more effectively to recipient organisms at a relatively low temperature (25°C) compared to the IncFII plasmid. Moreover, various other plasmid types were identified harboring blaNDM-1, including IncFIB, IncFII, IncL/M, and IncA/C2, among clinical isolates of Klebsiella pneumoniae or Enterobacter cloacae complex. Overall, our results highlight three distinct patterns of the dissemination of blaNDM-harboring plasmids among CPE isolates in Myanmar, contributing to a better understanding of their molecular epidemiology and dissemination in a setting of endemicity.

First Clinical Case of In Vivo Acquisition of DHA-1 Plasmid-Mediated AmpC in a Salmonella enterica subsp. enterica Isolate.

A pan-susceptible Salmonella enterica serovar Worthington isolate was detected in the stool of a man returning from Sri Lanka. Under ceftriaxone treatment, a third-generation cephalosporin (3GC)-resistant Salmonella Worthington was isolated after 8 days. Molecular analyses indicated that the two isolates were identical. However, the latter strain acquired a blaDHA-1-carrying IncFII plasmid probably from a Citrobacter amalonaticus isolate colonizing the gut. This is the first report of in vivo acquisition of plasmid-mediated resistance to 3GCs in S. enterica.

Genes and Proteins Involved in qnrS1 Induction.

Expression of the quinolone resistance gene qnrS1 is increased by quinolones, but unlike induction of some other qnr genes, the bacterial SOS system is not involved and no lexA box is found upstream. Nonetheless, at least 205 bp of upstream sequence is required for induction to take place. An upstream sequence bound to beads trapped potential binding proteins from cell extracts that were identified by mass spectrometry as Dps, Fis, Ihf, Lrp, CysB, and YjhU. To further elucidate their role, a reporter plasmid linking the qnrS1 upstream sequence to lacZ was introduced into cells of the Keio collection with single-gene deletions and screened for lacZ expression. Mutants in ihfA and ihfB had decreased lacZ induction, while induction in a cysB mutant was increased and dps, fis, lrp, yjhU, and other mutants showed no change. The essential upstream sequence contains potential binding sites for Ihf and DnaA. A dnaA deletion could not be tested because it provides essential functions in cell replication; however, increased dnaA expression decreased qnrS1 induction while decreased dnaA expression enhanced it, implying a role for DnaA as a repressor. In a mobility shift assay, purified IhfA, IhfB, and DnaA proteins (but not CysB) were shown to bind to the upstream segment. Induction decreased in a gyrA quinolone-resistant mutant, indicating that GyrA also has a role. Thus, quinolones acting through proteins DnaA, GyrA, IhfA, and IhfB regulate expression of qnrS1.

Enterobacter cloacae Complex Sequence Type 171 Isolates Expressing KPC-4 Carbapenemase Recovered from Canine Patients in Ohio.

Companion animals are likely relevant in the transmission of antimicrobial-resistant bacteria. Enterobacter xiangfangensis sequence type 171 (ST171), a clone that has been implicated in clusters of infections in humans, was isolated from two dogs with clinical disease in Ohio. The canine isolates contained IncHI2 plasmids encoding blaKPC-4 Whole-genome sequencing was used to put the canine isolates in phylogenetic context with available human ST171 sequences, as well as to characterize their blaKPC-4 plasmids.

Spread of Plasmid-Encoded NDM-1 and GES-5 Carbapenemases among Extensively Drug-Resistant and Pandrug-Resistant Clinical Enterobacteriaceae in Durban, South Africa.

Whole-genome sequence analyses revealed the presence of blaNDM-1 (n = 31), blaGES-5 (n = 8), blaOXA-232 (n = 1), or blaNDM-5 (n = 1) in extensively drug-resistant and pandrug-resistant Enterobacteriaceae organisms isolated from in-patients in 10 private hospitals (2012 to 2013) in Durban, South Africa. Two novel NDM-1-encoding plasmids from Klebsiella pneumoniae were circularized by PacBio sequencing. In p19-10_01 [IncFIB(K); 223.434 bp], blaNDM-1 was part of a Tn1548-like structure (16.276 bp) delineated by IS26 The multireplicon plasmid p18-43_01 [IncR_1/IncFIB(pB171)/IncFII(Yp); 212.326 bp] shared an 80-kb region with p19-10_01, not including the blaNDM-1-containing region. The two plasmids were used as references for tracing NDM-1-encoding plasmids in the other genome assemblies. The p19-10_01 sequence was detected in K. pneumoniae (n = 7) only, whereas p18-43_01 was tracked to K. pneumoniae (n = 4), Klebsiella michiganensis (n = 1), Serratia marcescens (n = 11), Enterobacter spp. (n = 7), and Citrobacter freundii (n = 1), revealing horizontal spread of this blaNDM-1-bearing plasmid structure. Global phylogeny showed clustering of the K. pneumoniae (18/20) isolates together with closely related carbapenemase-negative ST101 isolates from other geographical origins. The South African isolates were divided into three phylogenetic subbranches, where each group had distinct resistance and replicon profiles, carrying either p19-10_01, p18-10_01, or pCHE-A1 (8,201 bp). The latter plasmid carried blaGES-5 and aacA4 within an integron mobilization unit. Our findings imply independent plasmid acquisition followed by local dissemination. Additionally, we detected blaOXA-232 carried by pPKPN4 in K. pneumoniae (ST14) and blaNDM-5 contained by a pNDM-MGR194-like genetic structure in Escherichia coli (ST167), adding even more complexity to the multilayer molecular mechanisms behind nosocomial spread of carbapenem-resistant Enterobacteriaceae in Durban, South Africa.

CrpP Is a Novel Ciprofloxacin-Modifying Enzyme Encoded by the Pseudomonas aeruginosa pUM505 Plasmid.

The pUM505 plasmid, isolated from a clinical Pseudomonas aeruginosa isolate, confers resistance to ciprofloxacin (CIP) when transferred into the standard P. aeruginosa strain PAO1. CIP is an antibiotic of the quinolone family that is used to treat P. aeruginosa infections. In silico analysis, performed to identify CIP resistance genes, revealed that the 65-amino-acid product encoded by the orf131 gene in pUM505 displays 40% amino acid identity to the Mycobacterium smegmatis aminoglycoside phosphotransferase (an enzyme that phosphorylates and inactivates aminoglycoside antibiotics). We cloned orf131 (renamed crpP, for ciprofloxacin resistance protein, plasmid encoded) into the pUCP20 shuttle vector. The resulting recombinant plasmid, pUC-crpP, conferred resistance to CIP on Escherichia coli strain J53-3, suggesting that this gene encodes a protein involved in CIP resistance. Using coupled enzymatic analysis, we determined that the activity of CrpP on CIP is ATP dependent, while little activity against norfloxacin was detected, suggesting that CIP may undergo phosphorylation. Using a recombinant His-tagged CrpP protein and liquid chromatography-tandem mass spectrometry, we also showed that CIP was phosphorylated prior to its degradation. Thus, our findings demonstrate that CrpP, encoded on the pUM505 plasmid, represents a new mechanism of CIP resistance in P. aeruginosa, which involves phosphorylation of the antibiotic.