Comparative resistome and virulome analysis of clinical NDM-1-producing carbapenem-resistant Enterobacter cloacae complex.

OBJECTIVES: The Enterobacter cloacae complex (ECC) are causatives of hospital-acquired infections. The antimicrobial resistance (AMR) and virulence profiling of ECC promotes our knowledge for their elimination in clinical settings. METHODS: We assembled the whole genome of four clinical carbapenem-resistant ECCs and characterized their AMR and virulence profiles using whole genome sequencing. RESULTS: The chromosomes length scaled from minimum 3 949 952 bp (for P2) to maximum 4 976 575 bp (for P3). Strains P1 and P2 belonged to sequence type (ST)182. P3 and P4 belonged to ST477 and ST134, respectively. The blaCTX-M-15 gene was detected in P1 plasmid. P1 and P4 harboured the blaTEM-1 and blaOXA-1 genes. blaNDM-1 was found in P1, P3, and P4. No blaOXA-48, blaKPC, blaVIM, or blaIMP were identified. The plasmids were nontransferable and had IncFIB, IncFII, Col, and IncC incompatibility (Inc) groups . Class 1 integron was detected in all strains. Virulence genes related to biofilms, adhesins, siderophores (aerobactin, enterobactin, and salmochelin), intrinsic antimicrobial efflux pumps, secretory systems type I to VI, environmental and antibiotic stress response , outer membrane proteins, and heavy metal (copper, tellurite, arsenic, and zinc) resistance were found. The number of positive virulence factors was higher for P1 compared with other strains. CONCLUSION: The accumulation of AMR genes in Enterobacter spp. and their high endurance in hostile environments is a serious health problem. More genomic investigations are required to determine their AMR and virulence genetic reservoirs at the global level.

Comprehensive pan-genomic, resistome and virulome analysis of clinical OXA-48 producing carbapenem-resistant Serratia marcescens strains.

BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE) have been thoroughly studied as the pathogens associated with hospital acquired infections. However, data on Serratia marcescens are not enough. S. marcescens is now becoming a propensity for its highly antimicrobial-resistant clinical infections. METHODS: Four carbapenem-resistant S. marcescens (CR-SM) isolates were obtained from hospitalized patients through routine microbiological experiments. We assembled the isolates genomes using whole genome sequencing (WGS) and compared their resistome and virulome patterns. RESULTS: The average length and CG content of chromosomes was 5.33 Mbp and 59.8%, respectively. The number of coding sequences (CDSs) ranged from 4,959 to 4,989. All strains had one single putative conjugative plasmid with IncL incompatibility (Inc) group. The strains harbored blaCTX-M-15, blaTEM-1 and blaSHV-134. All plamsids were positive for blaOXA-48. No blaNDM-1, blaKPC, blaVIM and blaIMP were identified. The blaSRT-2 and aac(6')-Ic genes were chromosomally-encoded. Class 1 integron was detected in strains P8, P11 and P14. The Escher_RCS47 and Salmon_SJ46 prophages played major role in plasmid-mediated carraige of extended spectrum ß-lactamases (ESBLs). The CR-SM strains were equipt with typical virulence factors of oppotunistic pathogens including biofilm formation, adhesins, secretory systems and siderophores. The strains did not have ability to produce prodigiosin but were positive for chitinase and EstA. CONCLUSION: The presence of conjugative plasmids harboring major ß-lactamases within prophage and class 1 integron structures highlights the role of different mobile genetic elements (MGEs) in distribution of AMR factors and more specifically carbapenemases. More molecular studies are required to determine the status of carbapenem resistance in clinical starins. However, appropriate strategies to control the global dissemination of CR-SM are urgent.

High diversity of blaNDM-1-encoding plasmids in Klebsiella pneumoniae isolated from neonates in a Vietnamese hospital.

OBJECTIVES: The carbapenemase-encoding gene blaNDM-1 has been reported in Vietnam during the last 10 years, and blaNDM-producing Enterobacteriaceae are now silently and rapidly spreading. A key factor behind dissemination of blaNDM-1 is plasmids, mobile genetic elements that commonly carry antibiotic resistance genes and spread via conjugation. The diversity of blaNDM-1-encoding plasmids from neonates at a large Vietnamese hospital was characterized in this study. METHODS: 18 fecal Klebsiella pneumoniae and Klebsiella quasipneumoniae isolates collected from 16 neonates at a large pediatric hospital in Vietnam were studied using optical DNA mapping (ODM) and next-generation sequencing (NGS). Plasmids carrying the blaNDM-1 gene were identified by combining ODM with Cas9 restriction. The plasmids in the isolates were compared to investigate whether the same plasmid was present in different patients. RESULTS: Although the same plasmid was found in some isolates, ODM confirmed that there were at least 10 different plasmids encoding blaNDM-1 among the 18 isolates, thus indicating wide plasmid diversity. The ODM results concur with the NGS data. Interestingly, some isolates had two distinct plasmids encoding blaNDM-1 that could be readily identified with ODM. The coexistence of different plasmids carrying the same blaNDM-1 gene in a single isolate has rarely been reported, probably because of limitations in plasmid characterization techniques. CONCLUSIONS: The plasmids encoding the blaNDM-1 gene in this study cohort were diverse and may represent a similar picture in Vietnamese society. The study highlights important aspects of the usefulness of ODM for plasmid analysis.

Comparative genome analysis of colistin-resistant OXA-48-producing Klebsiella pneumoniae clinical strains isolated from two Iranian hospitals.

BACKGROUND: Carbapenemase-producing Klebsiella pneumoniae (CP-KP) is becoming extensively disseminated in Iranian medical centers. Colistin is among the few agents that retains its activity against CP-KP. However, the administration of colistin for treatment of carbapenem-resistant infections has increased resistance against this antibiotic. Therefore, the identification of genetic background of co-carbapenem, colistin-resistance K. pneumoniae (Co-CCRKp) is urgent for implementation of serious infection control strategies. METHODS: Fourteen Co-CCRKp strains obtained from routine microbiological examinations were subjected to molecular analysis of antimicrobial resistance (AMR) using whole genome sequencing (WGS). RESULTS: Nine of 14 K. pneumoniae strains belonged to sequence type (ST)-11 and 50% of the isolates had K-locus type 15. All strains carried blaOXA-48 except for P26. blaNDM-1 was detected in only two plasmids associated with P6 and P26 strains belonging to incompatibility (Inc) groups; IncFIB, IncHI1B and IncFII. No blaKPC, blaVIM and blaIMP were identified. Multi-drug resistant (MDR) conjugative plasmids were identified in strains P6, P31, P35, P38 and P40. MICcolistin of K. pneumoniae strains ranged from 4 to 32 µg/ml. Modification of PmrA, PmrB, PhoQ, RamA and CrrB regulators as well as MgrB was identified as the mechanism of colistin resistance in our isolates. Single amino acid polymorphysims (SAPs) in PhoQ (D150G) and PmrB (R256G) were identified in all strains except for P35 and P38. CrrB was absent in P37 and modified in P7 (A200E). Insertion of ISKpn72 (P32), establishment of stop codon (Q30*) (P35 and P38), nucleotides deletion (P37), and amino acid substitution at position 28 were identified in MgrB (P33 and P42). None of the isolates were positive for plasmid-mediated colistin resistance (mcr) genes. P35 and P38 strains carried iutA, iucD, iucC, iucB and iucA genes and are considered as MDR-hypervirulent strains. P6, P7 and P43 had ICEKp4 variant and ICEKp3 was identified in 78% of the strains with specific carriage in ST11. CONCLUSION: In our study, different genetic modifications in chromosomal coding regions of some regulator genes resulted in phenotypic resistance to colistin. However, the extra-chromosomal colistin resistance through mcr genes was not detected. Continuous genomic investigations need to be conducted to accurately depict the status of colistin resistance in clinical settings.

A Cross-Sectional Cohort Study of Extended-Spectrum-Beta-Lactamase-Producing Enterobacterales in Patients with Traveler's Diarrhea.

Patients with traveler's diarrhea (TD) can acquire extended-spectrum-beta-lactamase (ESBL)-producing members of the Enterobacterales (EPE) during travel to areas of endemicity. The aim of the present study was to investigate the prevalence and characteristics of EPE carriage in travelers from southern Sweden who were sampled for bacterial diagnostics of TD compared to those of EPE carriage 10 years ago. Clinical samples sent for culture of common causes of bacterial enterocolitis, if the referral stated foreign travel, were included in the study. Antimicrobial susceptibility testing was done according to the EUCAST disk diffusion test method. EPE strains were subjected to whole-genome sequencing (WGS). Eighty-four of 303 patients carried a total of 92 ESBL-producing members of the Enterobacterales The overall prevalence of EPE in tested samples was thus 28%, compared to 24% 10 years earlier (P = 0.33). Among 86 strains available for WGS, 47 different sequence types (STs) were identified, and there were only 5 ST131 strains. Of the 79 Escherichia coli isolates, 76% carried at least one fim (type 1 fimbria) gene, 29% carried at least one pap (p-fimbriae) gene, and 43% were extraintestinal pathogenic E. coli (ExPEC) or uropathogenic E. coli (UPEC). Over half of the E. coli strains (57%) were intestinal pathogenic E. coli, most commonly enteroaggregative E. coli (EAEC) (33%), and enteroinvasive E. coli EIEC (22%). A relatively high proportion of patients with traveler's diarrhea carry EPE, but there was no significant increase compared to 10 years ago. Most E. coli strains were intestinal pathogenic strains. A comparatively high proportion of the strains were ExPEC/UPEC, many expressing the virulence genes pap and/or fim (This project was assigned ClinicalTrials.gov number NCT03866291.).

Molecular Epidemiology of OXA-48 and NDM-1 Producing Enterobacterales Species at a University Hospital in Tehran, Iran, Between 2015 and 2016.

Carbapenem-resistant Enterobacterales (CRE) is an increasing problem worldwide. Here, we examined the clonal relatedness of 71 non-repetitive CRE isolates collected in a university hospital in Tehran, Iran, between February 2015 and March 2016. Pulsed-field gel electrophoresis (PFGE) and MLST were used for epidemiological analysis. Screening for antibiotic resistance genes, PCR-based replicon typing, conjugation experiments, and optical DNA mapping were also performed. Among all 71 isolates, 47 isolates of Klebsiella pneumoniae (66.2%), eight Escherichia coli (11.2%), five Serratia marcescens (7%), and two Enterobacter cloacae (2.8%) harbored bla NDM-1 and bla OXA-48 genes together or alone. PFGE analysis revealed that most of the OXA-48- and NDM-1-producing K. pneumoniae and all of OXA-48-producing S. marcescens were clonally related, while all eight E. coli and two E. cloacae isolates were clonally unrelated. The predominant clones of carbapenemase-producing K. pneumoniae associated with outbreaks within the hospital were ST147 (n = 13) and ST893 (n = 10). Plasmids carrying bla NDM-1 and bla OXA-48 were successfully transferred to an E. coli K12-recipient strain. The bla OXA-48 gene was located on an IncL/M conjugative plasmid, while the bla NDM-1 gene was located on both IncFII ∼86-kb to ∼140-kb and IncA/C conjugative plasmids. Our findings provide novel epidemiologic data on carbapenemase-producing Enterobacterales (CPE) in Iran and highlight the importance of horizontal gene transfer in the dissemination of bla NDM-1 and bla OXA-48 genes. The occurrence and transmission of distinct K. pneumoniae clones call for improved infection control to prevent further spread of these pathogens in Iran.

Successful international clones of blaCTX-M-15-producing Klebsiella pneumoniae with coexpression of plasmid-mediated quinolone resistance (PMQR) determinants in Tehran hospitals.

The dissemination of plasmid-mediated multidrug resistance in Enterobacteriaceae is a major public health concern. We investigated the prevalence of plasmid-mediated quinolone resistance (PMQR), 16S rRNA methylases, CTX-M, and acquired AmpC enzymes in ESBL-producing Klebsiella pneumoniae (n=40) from Tehran hospitals. Plasmid replicon typing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) were carried out for typing. CTX-M group 1 (confirmed as bla(CTX-M-15) in selected isolates) was found in 35/40 isolates. Thirty-two isolates hosted PMQR genes. Twenty isolates featured aac(6')-Ib-CR only; 9 isolates had aac(6')-Ib-CR and qnrB; 2 isolates had aac(6')-Ib-CR and qnrS; and 1 isolate had aac(6')-Ib-CR, qnrS, and qepA. The 16S rRNA methylase RmtB was found in 1 isolate; and acquired AmpC enzymes, in 6 isolates. PFGE detected 7 pulsotypes, the largest corresponded to sequence type 16. The successful clone ST101 was also found. The emergence of successful clones of K. pneumoniae in Tehran hospitals is concerning.

High prevalence of 16S rRNA methylase RmtB among CTX-M extended-spectrum ß-lactamase-producing Klebsiella pneumoniae from Islamabad, Pakistan.

The aim of this study was to characterise extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae isolated from urinary tract and wound infections from Pakistan (n=25). Isolates were subjected to commercially available microarray analysis to determine the presence of ESBLs and acquired AmpC enzymes. The genetic diversity of the isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmid replicon typing and capsular serotyping were conducted by PCR. Finally, screening for virulence genes, plasmid-mediated quinolone resistance (PMQR) genes, and genes encoding 16S rRNA methylases was done using PCR. All K. pneumoniae isolates hosted blaCTX-M genes and all strains belonged to phylogroup CTX-M-1. Acquired AmpC ß-lactamases (ACT/MIR and CIT group) were found in 16% of isolates. Two clusters were observed with ≥80% similarity among profiles obtained by PFGE, and two sequence types (STs) by MLST, namely ST215 and ST307, were observed in these clusters. Three ST215 isolates carried virulence factor wcaG and three ST215 isolates had capsular type K20. IncFIA, IncFIB, IncFIIK and FrepB replicons were most commonly found in this collection. Among the PMQR determinants, aac(6')-lb-cr was present in 96% (24/25) of the isolates, qnrB was found in 88% (22/25) and qepA was found in 4% (1/25). The 16S rRNA methylase-encoding gene rmtB was found in 60% (15/25) of the isolates. In conclusion, CTX-M-producing ST215 and ST307 K. pneumoniae were the two major clones detected. Of particular concern was the high prevalence of 16S rRNA methylases conferring resistance to all aminoglycosides.

Molecular characterization of CTX-Mß-lactamases among Klebsiella pneumoniae isolated from patients at Tehran hospitals.

PURPOSE: Plasmid-encoded CTX-M-group of extended-spectrum ß-lactamases (ESBLs) represent a significant and rapidly emerging problem in most part of the world. The aim of the present study was to describe the prevalence of CTX-M producing Klebsiella pneumoniae at Tehran hospitals. MATERIALS AND METHODS: Clinical isolates of K. pneumoniae (n=250) were collected from 10 hospitals of Tehran. Susceptibility to antimicrobial agents, MIC of cefotaxime and ESBLs production of collected isolates were detected. All ESBL-producing isolates were screened for bla CTX-M genes using PCR and DNA sequencing. Molecular typing of bla(CTX-M) harboring isolates was performed by Pulsed-field gel electrophoresis assay. RESULTS: Of 250 K. pneumoniae clinical isolates, 102 isolates revealed ESBLs - phenotype. PCR assay and sequencing detected bla(CTX-M) genes in 71.5% (n= 73) of ESBL-producing isolates. The prevalence of CTX-M -I and CTX-M-III clusters among these isolates was 35.61% (n=26) and 21.9 % (n=16) respectively. Coexistence of CTX-M -I and CTX-M-III clusters was found among 42.5% (n= 31) of isolates. Of 102 isolates that were positive in the phenotypic confirmatory test (PCT), 29 isolates (28.4%) did not produce any amplicons in PCR for bla(CTX-M) gene. The results of PCR for CTX-M -II and CTX-M-IV clusters were also negative. Analysis of the 31 CTX-M producing K. pneumoniae isolates by PFGE typing showed 26 distinct patterns. CONCLUSIONS: The bla CTX-M genes are widespread among Iranian isolates of K. pneumoniae. PFGE demonstrated the high diversity of K. pneumoniae harboring bla(CTX-M) in our study.