Mediated Carbapenem Resistance Research Articles

Antimicrobial Resistance and Genomic Characteristics of Escherichia coli Strains Isolated from the Poultry Industry in Henan Province, China.

Escherichia coli (E. coli) is an important foodborne pathogen and a biomarker for monitoring antimicrobial resistance. Investigating the prevalence of E. coli in the poultry industry holds great importance, particularly in Henan province, a major poultry-producing region in China. Here, we investigated the antimicrobial resistance (AMR) phenotypes of E. coli strains obtained from the poultry industry in Henan, China. A total of 344 E. coli strains were isolated from 638 samples collected from seven farms, three slaughterhouses, and ten terminal markets. Approximately 96.4%, 81.7%, and 52.5% of the isolates from the farms, slaughterhouses, and terminal markets exhibited multidrug resistance. Whole-genome sequencing was performed on 169 strains to reveal their genomic characteristics. The sequence type (ST) analysis revealed that ST10 and ST156 were the most frequent types within the poultry supply chain, whereas ST10 and ST162 were commonly found across the farms, slaughterhouses, and terminal markets. Fourteen ST10 E. coli strains belonged to phylogenetic group A, while fifteen ST165 and six ST162 E. coli strains belonged to phylogenetic group B1. In addition, several antimicrobial resistance genes and virulence factor genes were identified. The blaNDM-5 gene mediated carbapenem resistance in two E. coli strains, while mcr-1-mediated colistin resistance was detected in nine E. coli strains. Phylogenetic group A exhibited fewer virulence genes compared to other groups of E. coli. Plasmid replicons, such as IncFIB (AP001918), IncX1, IncFIC (FII), and IncFII (pHN7A8), were frequently observed. These findings provide valuable insights into the current AMR profiles of E. coli strains isolated from the poultry industry in Central China and highlight the need to implement good manufacturing practices and reduce antibiotic usage to mitigate potential risks associated with E. coli.

Molecular characterization of IncFII plasmid carrying blaNDM-5 in a Salmonella enterica serovar Typhimurium ST34 clinical isolate in China.

In this study, an IncFII plasmid pIncFII-NDM5 carrying blaNDM-5 was found in carbapenem-resistant Salmonella enterica serovar Typhimurium (S. enterica serovar Typhimurium), which has conjugative transferability and carried blaNDM-5, bleMBL, mph(A), and blaTEM-1 four resistance genes that can mediate resistance to multiple antibiotics including cephalosporins, beta-lactamase inhibitor combinations, carbapenems, and macrolides. Phylogenetic analysis showed that 1104-65 and 1104-75 were closely related to other S. enterica serovar Typhimurium in this area. The above-mentioned S. enterica serovar Typhimurium chromosome carries blaCTX-M-55, qnrS1, and tet(A) genes, so the antibiotic resistance of isolates will be further enhanced after obtaining the pIncFII_NDM5-like plasmid. Meanwhile, we discovered a novel genetic structure of blaNDM-5 mediated by the IS26 composite transposon, which will expand our understanding of the emergence and spread of carbapenem-resistance genes. Altogether, the presence of the IncFII plasmid pIncFII-NDM5 further underscores the need for vigilant surveillance and appropriate infection control measures to mitigate the impact of carbapenem-resistant S. enterica serovar Typhimurium in clinical settings.

Factors associated with non-carbapenemase mediated carbapenem resistance of Gram-negative bacteria: a retrospective case-control study

Infections with carbapenemase-producing Gram-negative bacteria are related to increased morbidity and mortality, yet little is known regarding infections caused by non-beta-lactamase mediated carbapenem-resistant bacteria. Our objective was to identify risk factors for, and the clinical impact of infections caused by carbapenem-resistant carbapenemase-negative Enterobacterales and Pseudomonas aeruginosa. This retrospective matched case-control study was performed at the University Hospital of Basel, Switzerland, in 2016. We focused on other resistance mechanisms by excluding laboratory-confirmed carbapenemase-positive cases. Carbapenem resistance was set as the primary endpoint, and important risk factors were investigated by conditional logistic regression. The clinical impact of carbapenem resistance was estimated using regression models containing the resistance indicator as explanatory factor and adjusting for potential confounders. Seventy-five cases of infections with carbapenem-resistant, carbapenemase-negative bacteria were identified and matched with 75 controls with carbapenem-susceptible infections. The matched data set was well-balanced regarding age, gender, and comorbidity. Duration of prior carbapenem treatment (OR 1.15, [1.01, 1.31]) correlated with resistance to carbapenems. Our study showed that patients with carbapenem-resistant bacteria stayed 1.59 times (CI [0.81, 3.14]) longer in an ICU. The analyzed dataset did not provide evidence for strong clinical implications of resistance to carbapenems or increased mortality. The duration of prior carbapenem treatment seems to be a strong risk factor for the development of carbapenem resistance. The higher risk for a longer ICU stay could be a consequence of a carbapenem resistance. In contrast to carbapenemase-producers, the clinical impact of carbapenamase-negative, carbapenem-resistant strains may be limited. Trial registration: The study design was prospectively approved by the local Ethics Commission on 10.08.2017 (EKNZ BASEC 2017-00222).

Potentiate the activity of current antibiotics by naringin dihydrochalcone targeting the AdeABC efflux pump of multidrug-resistant Acinetobacter baumannii

Acinetobacter baumannii is an ESKAPE pathogen responsible for severe nosocomial infections. Among all the mechanisms contributing to multidrug resistance, efflux pumps have gained significant attention due to their widespread distribution among bacterial species and broad substrate specificity. This study has investigated the diverse roles of efflux pumps present in carbapenem-resistant A. baumannii (CRAB) and screen an efflux pump inhibitor. The result showed the presence of AdeABC, AdeFGH, AdeIJK, and AbeM efflux pumps in CRAB, and experimental studies using gene mutants demonstrated the significant role of AdeABC in carbapenem resistance, biofilm formation, surface motility, pathogenesis, bacterial adherence, and invasion to the host cells. The structure-based ligand screening, molecular mechanics, molecular dynamics simulation, and experimental validation using efflux pump mutants and antibiotic accumulation assay identified naringin dihydrochalcone (NDC) as the lead against AdeB. This lead was selected as a capping agent for silver nanoparticles. The NDC-capped silver nanoparticles (NDC-AgNPs) were characterized by UV-spectroscopy, Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), and scanning electron microscopy (SEM). The investigated molecular mechanism showed that the NDC-AgNPs possessed multiple mechanisms of action. In addition to efflux inhibitory activity, it also generates reactive oxygen and nitrogen species as well as causes change in the electrochemical gradient in CRAB. The proton gradient is important for the function of AdeABC; hence altering the electrochemical gradient also disrupts its efflux activity. Moreover, A. baumannii did not develop any resistance against NDC-AgNPs till several generations which were investigated. The NDC-AgNPs were also found to be effective against carbapenem-resistant clinical isolates of A. baumannii. Therefore, the present study provided an insight into the efflux pump mediated carbapenem resistance and possible inhibitor NDC-AgNPs to combat AdeABC efflux pump mediated resistance.

English

Bacteria develop antimicrobial drug resistance through several mechanisms, the common one being the production of enzymes. As the number of antibiotics discovered is in notable numbers in the past few years, it is important to preserve high-end antibiotics for the treatment of multidrug-resistant organisms (MDROs) infections, by appropriate use of antibiotics. A study was conducted to record prevalence, phenotypic and genotypic characters of MDROs in our hospital, with reference to carbapenem resistance. 200 multidrug-resistant clinical isolates were collected in 6 months. Carbapenem-resistant organisms were detected phenotypically confirmed for the production of carbapenemases by modified Hodge test (MHT) and genotypic detection was done by a multiplex polymerase chain reaction (PCR) assay for the five most predominant carbapenemases (bla NDM-1, bla OXA-48 , bla VIM, bla IMP, bla KPC). The isolates consisted of E. coli (53 %) followed by K. pneumoniae (30 %), P. aeruginosa (13 %), and acinetobacter spp (4 %). Among these, 40 (20 %) isolates were carbapenem-resistant. Of these 40, 27 (67.5 %) showed an increase in zone size by the MHT, suggestive of metallo-beta-lactamase (MBL) mediated carbapenem resistance and about 32 (80 %) isolates were found to contain at least one carbapenemase gene. bla NDM-1 accounted for 37.5 % (12 / 32) of the isolates and was the most predominant one followed by bla OXA-48 [28 % (9 / 32)]. 22 % (7 / 32) of the isolates had one or more carbapenemase genes. Identifying the mechanisms of resistance of pathogens is important to implement strict infection prevention and control measures in the hospital to prevent the transmission of the resistant pathogens. KEY WORDS Multidrug-Resistant Bacteria, Bla NDM-1 Gene, Bla OXA-48 Gene, Carbapenem Resistance, Carbapenem Resistant Organisms.

Nosocomial infection by Klebsiella pneumoniae among neonates: a molecular epidemiological study

Nosocomial infection by Klebsiella pneumoniae (Kp) and drug resistance of Kp among neonates is a major concern. Hypervirulent K.pneumoniae (hvKp) infections are gradually increasing worldwide. Carbapenem-resistant hvKp infection has brought challenges to clinical treatment. To evaluate the changes in drug resistance trends of Kp strains in neonatal intensive care unit (NICU) nosocomial infections, to analyse drug resistance genes and virulence genes of carbapenem-resistant K.pneumoniae (CRKP) and to identify whether these CRKP strains are hvKp. A total of 80 neonates with Kp nosocomial infections from 2013 to 2018 were retrospectively studied. Drug susceptibility testing was performed on 80 Kp strains, among which the 12 CRKP strains were further studied. Kp accounted for 26.9% of nosocomial infections in the NICU. CRKP strains accounted for 15.0%. Among the 80 nosocomial infection Kp strains, CRKP strains accounted for 33.3% and 53.3% in 2017 and 2018 respectively. One of the 12 CRKP strains was positive in the drawing test. The 12 CRKP strains were divided into four complete genome sequence types: cgST1 (N= 2), cgST2 (N= 1), cgST3 (N= 1), and cgST4 (N= 8). Among genes that mediated carbapenem resistance, strains of cgST4 carried NDM-5, strains of cgST2 and cgST3 carried NDM-1, and strains of cgST1 carried IMP-4. None of the 12 CRKP strains carried rmpA/rmpA2 (highly related with hvKp). Nosocomial infections of CRKP among neonates are becoming common, but no hvKp was found among the CRKP strains in this study.

Characterization of plasmids mediating carbapenem-resistance in Klebsiella pneumoniae in Pretoria, South Africa

Background: Mobile genetic elements such as plasmids play a major role in the acquisition and dissemination of antimicrobial resistance determinants in carbapenem-resistant Klebsiella pneumoniae (CRKP). This study aims to determine the frequency of plasmids replicon groups and characterize plasmids mediating carbapenem resistance in K. pneumoniae isolates in South Africa. Methods and materials: A total of 56 K. pneumoniae isolates already identified by the national referral laboratory in Pretoria using the VITEK 2® (Biomerieux, France) automated system. Antimicrobial susceptibility testing was performed using the MicroScan Gram-negative MIC 44 panel (Beckman Coulter, United States). All K. pneumoniae resistant to one or more carbapenem(s) were screened for carbapenemase-encoding genes (blaOXA-48, blaNDM-1, blaKPC, blaVIM, and blaIMP) using multiplex-PCR. These isolates were genotyped by REP-PCR. Plasmid extraction was performed on all isolates and electrophoresis was used to determine their number and size. PCR-based replicon typing (PBRT) scheme was used to determine the incompatibility/replicon groups of all the extracted plasmids. Results: The isolates showed reduced susceptibility to tested carbapenems. Multiplex-PCR analysis showed that 55 isolates haboured at least one of the carbapenemase genes, with 41 (73.2%) habouring blaOXA-48 and 18 (32%) habouring blaNDM-1. The isolates were resolved into four major strains/genotypes by the REP-PCR. Electrophoresis revealed that the isolates carried between one and five plasmids, with the majority carrying 2 or 3 plasmids; the plasmid sizes ranged between 1.6-kb to >48.5-kb. IncF (FII, FIB, FIC), IncL, IncM, and IncA/C plasmid replicons were the most detected. Almost 90% of the isolates showed multi-replicon carriage (Table 1).Tabled 1Isolate NoCarbapenemase genesPlasmid NoSize estimation (kb)Plasmid IncStrainKp15OXA-4851.7–≥48.5FIB, A/C,FIIk1Kp10NDM-1, OXA-4842.4–≥48.5HI2, M, FIIk2Kp37NDM-1, OXA-4842.4–≥48.5L, M, FII, FIIk3Kp12OXA-482≥48.5M, FIIk4 Open table in a new tab Conclusion: This study shows an ongoing plasmid-mediated endemicity/dissemination of CRKP in the Gauteng province, with blaOXA-48 and blaNDM-1-producing CRKP being predominant in 2018. Rigorous infection prevention and control, including contact precautions, should be adopted in all Gauteng hospitals to curtail further escalation of CRKP.

Higher Prevalence of Multi-Antimicrobial Resistant Bacteroides spp. Strains Isolated at a Tertiary Teaching Hospital in China.

PurposeThe study investigates the molecular epidemiology of multi-drug resistant (MDR) Bacteroides spp. isolates and the clinical characteristics of the patients.Materials and MethodsBacteroides spp. clinical strains were identified through MALDI-TOF MS and VITEK-2 anaerobes and corynebacterium (ANC) cards. A broth microdilution method was employed to detect the antimicrobial sensitivities of Bacteroides spp. isolates. PCR was used to detect the resistance genes, including cfxA, cepA, cfiA, ermF, nim, as well as the upstream insertion sequence (IS) element of the cfiA gene. The effects of broad-spectrum efflux pump inhibitors (EPIs) on the minimal inhibitory concentration (MICs) of cefoxitin, moxifloxacin, and imipenem for MDR Bacteroides spp. were investigated.ResultsThe total resistance rates of 115 Bacteroides spp. isolates to cefoxitin, moxifloxacin, clindamycin, metronidazole, imipenem and meropenem were 4.3%, 16.5%, 80.0%, 5.2%, 13.9% and 13.9%, respectively. The positive rates of carbapenem resistance gene cfiA were 38.9% and 8.6% for B. fragilis and non-B. fragilis isolates, respectively. The isolation rate of MDR isolates reached up to 18.26% (21/115), and the isolation rate among the gastrointestinal cancer patients was significantly higher when compared to the non-gastrointestinal cancer patients (52.38%/26.08%, P = 0.006). Furthermore, MDR isolates were more likely to be isolated from the patients exposed to cephalosporins 3 months before Bacteroides spp. isolation (76.19%/31.52%, P = 0.000).ConclusionThe overall resistance rates of Bacteroides spp. isolates against multiple antimicrobials were at a high level, especially for B. fragilis. The CfiA gene carrying rate among B. fragilis isolates was as high as 38.9%, and its mediated carbapenem resistance was the major resistance mechanism for B. fragilis. The findings of this study imply that the real resistance tendency of Bacteroides spp. may be underestimated and need to be given more attention.

Role of plasmid carrying bla NDM in mediating antibiotic resistance among Acinetobacter baumannii clinical isolates from Egypt.

We investigated antibiotic resistance levels among blaNDM-positive (n = 9) and -negative (n = 65) A. baumannii clinical isolates collected in 2010 and 2015 from Alexandria Main University Hospital, Egypt using disc diffusion and minimum inhibitory concentration (MIC) determination. Plasmids from blaNDM-positive isolates were transformed into a carbapenem-susceptible A. baumannii (CS-AB) isolate to assess the role of plasmid transfer in mediating carbapenem resistance. Imipenem, meropenem, and ertapenem MIC90 values against blaNDM-positive isolates were 128, > 256, and 256 µg/mL, respectively. Plasmid isolation and polymerase chain reaction revealed that blaNDM was plasmid mediated. The plasmids were electroporated into the cells of a CS-AB isolate at an efficiency of 1.3 × 10–8 to 2.6 × 10–7, transforming them to blaNDM-positive carbapenem-resistant cells with an imipenem MIC increase of 256-fold. In addition to carbapenem resistance, the blaNDM-positive isolates also exhibited higher levels of cephalosporins, tetracycline, aminoglycosides, fluoroquinolones, and colistin resistance than the blaNDM-negative isolates. Acquisition of blaNDM-carrying plasmids dramatically increased imipenem resistance among A. baumannii isolates. Intriguingly, blaNDM-positive isolates also showed a high degree of resistance to antibiotics of different classes. The potential co-existence of different resistance determinants on A. baumannii plasmids and their possible transfer owing to the natural competence of the pathogen are especially alarming. More effective infection control and antibiotic stewardship programs are needed to curb the spread and treat such infections in both hospital and community settings.Electronic supplementary materialThe online version of this article (10.1007/s13205-020-2157-y) contains supplementary material, which is available to authorized users.

Characterization of a Carbapenem-Resistant Kluyvera Cryocrescens Isolate Carrying Blandm-1 from Hospital Sewage.

Carbapenem-resistant Enterobacteriaceae have been a global public health issue in recent years. Here, a carbapenem-resistant Kluyvera cryocrescens strain SCW13 was isolated from hospital sewage, and was then subjected to whole-genome sequencing (WGS). Based on WGS data, antimicrobial resistance genes were identified. Resistance plasmids were completely circularized and further bioinformatics analyses of plasmids were performed. A conjugation assay was performed to identify a self-transmissible plasmid mediating carbapenem resistance. A phylogenetic tree was constructed based on the core genome of publicly available Kluyvera strains. The isolate SCW13 exhibited resistance to cephalosporin and carbapenem. blaNDM-1 was found to be located on a ~53-kb self-transmissible IncX3 plasmid, which exhibited high similarity to the previously reported pNDM-HN380, which is an epidemic blaNDM-1-carrying IncX3 plasmid. Further, we found that SCW13 contained a chromosomal blaKLUC-2 gene, which was the probable origin of the plasmid-born blaKLUC-2 found in Enterobacter cloacae. Phylogenetic analysis showed that K. cryocrescens SCW13 exhibited a close relationship with K. cryocrescens NCTC10483. These findings highlight the further dissemination of blaNDM through clonal IncX3 plasmids related to pNDM-HN380 among uncommon Enterobacteriaceae strains, including Kluyvera in this case.

Coexistence of three blaKPC-2 genes on an IncF/IncR plasmid in ST11 Klebsiella pneumoniae.

Here we report the finding of three copies of the blaKPC-2 gene on a plasmid in ST11 Klebsiella pneumoniae. Carbapenem-resistant K. pneumoniae clinical strain WCHKP2 was subjected to whole-genome sequencing (WGS) using both a short-read Illumina HiSeq X10 platform and long-read MinION sequencer. Hybrid assembly was performed using Unicycler, and contigs were corrected using Pilon. Based on WGS, the sequence type (ST), capsular type, plasmid replicon type and plasmid multilocus sequence type were determined and virulence and antimicrobial resistance genes were identified. Mating was performed to identify a self-transmissible plasmid mediating carbapenem resistance. Strain WCHKP2 was resistant to imipenem [minimum inhibitory concentration (MIC)=64μg/mL] and meropenem (MIC=128μg/mL). Strain WCHKP2 had a 5477148-bp circular chromosome, two small ColRNAI-like plasmids (5596bp and 10060bp), and one large plasmid (177516bp, designated pKPC2_020002) containing an IncR and an IncFII replicon. Surprisingly, there were three copies of the blaKPC-2 carbapenemase gene on pKPC2_020002, which was not self-transmissible. Each of the blaKPC-2 genes was located in the same genetic context with insertion sequence ISKpn27 upstream and ISKpn6 downstream and bracketed by IS26. The three copies of the IS26-ISKpn27-blaKPC-2-ISKpn6-IS26 unit were present in tandem. Here we report the surprising co-existence of three copies of blaKPC-2 on an IncR/IncF plasmid due to the action of IS26. Multiple copies of IS26 are a key factor generating genetic plasticity and could mediate the multiplication of resistance genes.

New Delhi Metallo-β-lactamase (NDM)-mediated Carbapenem-Resistant Pseudomonas aeruginosa Clinical Isolates in Sudan

Carbapenem resistance mediated by NDM is particularly gruesome as this carbapenemase can hydrolyze a wide range of β-lactam antibiotics.
 Aim: This study aims to detect NDM mediated carbapenem resistance in clinical isolates of Pseudomonas aeruginosa.
 Materials and Methods: 50 multi-drug resistant clinical urinary isolates of Pseudomonas aeruginosa from three major hospitals in Khartoum state Sudan; Khartoum Teaching Hospital, Medical Army Hospital and Omdurman teaching hospital, in period from July 2016 to September 2017, were investigated for carbapenem resistance using standard disc diffusion method and underwent real-time PCR to detect carbapenem resistance gene blaNDM. Data were analyzed using IBM SPSS.
 Results: 60% were positive for the blaNDM, 82% were resistant to Imipenem and 75% of the samples were resistant to Meropenem.
 Conclusion: The emergence of carbapenem resistance is a global problem that requires earnest attention. To make the suitable preventive measures, the emergence of these genes must be monitored closely. Our findings revealed that carbapenem-resistant due to the gene blaNDM is accounted for 60% of the cases, and due to lack of proper data documentation about the emergence of this gene in Sudan, these cases to the best of our knowledge are the first to be reported in Sudan.

New Delhi Metallo-Beta-Lactamase-Producing Enterobacteriaceae in South Korea Between 2010 and 2015.

This study was carried out to investigate the epidemiological time-course of New Delhi metallo-beta-lactamase- (NDM-) mediated carbapenem resistance in Enterobacteriaceae in South Korea. A total of 146 non-duplicate NDM-producing Enterobacteriaceae recovered between 2010 and 2015 were voluntarily collected from 33 general hospitals and confirmed by PCR. The species were identified by sequences of the 16S rDNA. Antimicrobial susceptibility was determined either by the disk diffusion method or by broth microdilution, and the carbapenem MICs were determined by agar dilution. Then, multilocus sequence typing and PCR-based replicon typing was carried out. Co-carried genes for drug resistance were identified by PCR and sequencing. The entire genomes of eight random selected NDM producers were sequenced. A total of 69 Klebsiella pneumoniae of 12 sequence types (STs), 34 Escherichia coli of 15 STs, 28 Enterobacter spp. (including one Enterobacter aerogenes), nine Citrobacter freundii, four Raoultella spp., and two Klebsiella oxytoca isolates produced either NDM-1 (n = 126), NDM-5 (n = 18), or NDM-7 (n = 2). The isolates co-produced CTX-M-type ESBL (52.1%), AmpCs (27.4%), additional carbapenemases (7.1%), and/or 16S rRNA methyltransferases (4.8%), resulting in multidrug-resistance (47.9%) or extensively drug-resistance (52.1%). Among plasmids harboring blaNDM, IncX3 was predominant (77.4%), followed by the IncFII type (5.8%). Genome analysis revealed inter-species and inter-strain horizontal gene transfer of the plasmid. Both clonal dissemination and plasmid transfer contributed to the wide dissemination of NDM producers in South Korea.

Prevalence and Molecular Characterization of New Delhi Metallo-Beta-Lactamases in Multidrug-Resistant Pseudomonas aeruginosa and Acinetobacter baumannii from India.

New Delhi metallo-beta-lactamase (NDM)-mediated carbapenem resistance in Pseudomonas aeruginosa and Acinetobacter baumannii is a major concern. We investigated the presence of NDM and its variants in P. aeruginosa and A. baumannii at a tertiary hospital in North India. A total of 236 isolates (130 P. aeruginosa and 106 A. baumannii) were included; 38 (29.23%) P. aeruginosa and 20 A. baumannii isolates (18.8%) were resistant to carbapenems and all of them were blaNDM positive. All 38 carbapenem-resistant P. aeruginosa harbored blaNDM-1, while 12 (60%) of 20 A. baumannii harbored blaNDM-2. Pulsed-field gel electrophoresis showed that all 58 isolates were clonally unrelated. By Southern blot analysis, blaNDM-2 was located on chromosome. The blaNDM-2-positive isolates were more frequently recovered from tracheal aspirate (67% vs.16%; p = 0.02) and intensive care unit (67% vs. 20%; p = 0.001) than blaNDM-1. Among other carbapenemases, VIM was significantly associated with blaNDM-1 than blaNDM-2 (61% vs. 17%; p = 0.006). Mortality between blaNDM-1- and blaNDM-2-infected patients was comparable. When expressed in Escherichia coli, blaNDM-2 transformant conferred one doubling dilution higher MIC value for cefotaxime, piperacillin/tazobactam than blaNDM-1. The study shows the emergence of blaNDM-mediated resistance among P. aeruginosa and A. baumannii and rapid evolution of blaNDM-2 in A. baumannii with its chromosomal localization.

First report of blaOXA-181-mediated carbapenem resistance in Aeromonas caviae in association with pKP3-A: Threat for rapid dissemination.

Carbapenemase-producing Aeromonas spp. are of great concern in healthcare settings and are also known to acquire clinically relevant resistance genes. In this study, carbapenem-non-susceptible Aeromonas isolates were characterised for their molecular mechanisms of resistance. Among 180 Aeromonas isolates, 10 carbapenem-non-susceptible isolates were selected based on their antimicrobial susceptibility profile. Carbapenemase production was investigated by the CarbaNP test. ESBL-, AmpC- and carbapenemase-encoding genes were screened by PCR. Isolates VBF557 and VBF856 with high MICs for imipenem were selected for whole-genome sequencing (WGS). Conjugation experiments were performed to determine the transmissibility of resistance. WGS remarkably revealed the presence of class D β-lactamases (AmpS/AmpH), class C β-lactamases and class B2 metallo-β-lactamase (cphA3) in VBF557. In contrast, VBF856 had multiple resistance genes coding for aminoglycoside, sulphonamide, carbapenem (blaOXA-181 class D β-lactamase), macrolide, fluoroquinolone, rifampicin, phenicol, tetracycline and trimethoprim resistance. This is the first global report of blaOXA-181 in Aeromonas spp. Interestingly, blaOXA-181 was identified in association with transposon Tn2013 in plasmid pKP3-A. Additionally, an IncQ2 plasmid with qnrS2 was identified. Among the tested isolates, VBF1116 and VBF888 possessed blaNDM and blaVEB, respectively, by PCR. None of the other isolates harboured any tested β-lactamase genes. The resistance gene was transmissible in the presence of imipenem. Presence of such resistance genes in plasmids further adds complexity for control of spread of carbapenem resistance. This study reveals the emergence of carbapenem resistance among Aeromonas spp. and the importance of mobile genetic elements such as plasmids in interchanging resistance determinants between species.

Detection of ISAba1 in association with a novel allelic variant of the β-lactamase ADC-82 and class D β-lactamase genes mediating carbapenem resistance among the clinical isolates of MDR A. baumannii.

The objective of the present study is to investigate the diverse resistance determinants, their association with insertion sequence mobile elements and predilection of a particular clone for such associations in Acinetobacter baumannii. Fifty-four consecutive isolates collected during 2011-2012 from a tertiary care hospital were subjected to susceptibility testing followed by PCR screening of commonly reported β-lactamases and 16S rRNA methyltransferase encoding genes. The integrity of resistance-nodulation-cell division efflux pump-related genes in their respective operons was also investigated. β-Lactamase genes such as blaADC (100 %), blaOXA-23 (81 %), blaPER-1 (81 %), blaIMP-1 (31 %) and blaNDM-1 (15 %) were found to be present more frequently while blaVIM-2 and blaOXA-24 were not observed in our study population. ISAba1 was associated only with blaOXA-51-like like (30 %), blaOXA-23-like (55 %) and blaADC-like (33 %). armA was found in 87 % of isolates and ISAba1 linked with one novel variant of ADC, namely blaADC-82, which was identified to have 15 nucleotide differences with blaADC-79, and this finding is of much significance. In many isolates, efflux pump genes were not intact, resulting in severely altered effluxing functions. For the first time, we have identified ISAba1-mediated disruption of adeN among the isolates of ST 195B, which would have led to overexpression of AdeIJK efflux pump causing elevated resistance. Multilocus sequence typing revealed the predominance of CC 92B (IC-IIB) and CC 447B clonal complexes. High incidence of IC-II clones, novel resistance determinants (ADC-82) and elevated resistance mediated by ISAba1 reported here will be of enormous importance while assessing the emergence of extremely resistant A. baumannii in India.

Detection and termination of an extended low-frequency hospital outbreak of GIM-1–producing Pseudomonas aeruginosa ST111 in Germany

Metallo-β-lactamase German imipenemase-1 (GIM-1)-mediated carbapenem resistance is emerging in Germany but has not spread beyond a very localized region. The aim of this study was to describe the first outbreak of an extensively drug-resistant GIM-1-carrying Pseudomonas aeruginosa strain affecting 29 patients in a tertiary care hospital from 2002-2013. The outbreak was studied retrospectively and prospectively by a combination of molecular methods (carbapenemase polymerase chain reaction [PCR]), genotyping (DiversiLab, pulsed field gel electrophoresis and multi-locus sequence typing, bioMérieux, Marcy l'Etoile, France), descriptive epidemiology, and extensive environmental investigations using swabs with liquid transport medium, blaGIM-1 PCR, directly from the medium and culture. Of the 29 affected patients, 24 had been admitted to a surgical intensive care unit at some point, where environmental sampling revealed a high burden of blaGIM-1 in the wastewater system. The outbreak strain was found in several sinks and on a reusable hair washbasin. Initially, general infection control measures were applied; thereafter, specific measures were implemented, including the restriction of washbasin use. Continued surveillance over a period of 2 years has revealed no further case of GIM-1-carrying Pseudomonas aeruginosa. This long-term outbreak highlights the potential of molecular methods in surveillance for multidrug-resistant pathogens and in environmental sampling and the successful containment by application of specific control measures targeting biofilms within sink drains as potential environmental reservoirs for P aeruginosa.

Emergence of integron borne PER-1 mediated extended spectrum cephalosporin resistance among nosocomial isolates of Gram-negative bacilli.

Background & objectives:Pseudomonas extended resistant (PER) enzymes are rare type of extended-spectrum beta lactamases (ESBLs) that confer third generation cephalosporin resistance. These are often integron borne and laterally transmitted. The aim of the present study was to investigate the emergence of integron borne cephalosporin resistant PER-1 gene in diverse incompatibility (Inc) group plasmids among Gram-negative bacteria.Methods:A total of 613 consecutive, non-duplicate, Gram-negative bacteria of Enterobacteriaceae family and non-fermenting Gram-negative bacteria were isolated from different clinical specimens during a period of 18 months. For amplification and detection of blaPER, multiplex PCR was done. For understanding the genetic environment of blaPER-1, integrase gene PCR and cassette PCR (59 be) was performed. Gene transferability experiment was carried out and PCR based replicon typing was performed for incompatibility group typing of plasmids using 18 pairs of primers. An inhibitor based method was used for phenotypic detection of intrinsic resistance.Results:Multiplex PCR and sequencing confirmed that 45 isolates were harbouring blaPER-1. Both class 1 and class 2 integrons were observed among them. Integrase and cassette PCR (59 be) PCR results confirmed that the resistant determinant was located within class 1 integron. Transformation and conjugation experiments revealed that PER-1 was laterally transferable and disseminated through diverse Inc plasmid type. Efflux pump mediated carbapenem resistance was observed in all isolates. All isolates belonged to heterogenous groups.Interpretation & conclusions:This study demonstrates the dissemination of cephalosporins resistant, integron borne blaPER-1 in hospital setting in this part of the country and emphasizes on the rational use of third generation cephalosporins to slow down the expansion of this rare type of ESBL gene.

Prevalence and molecular characterisation of New Delhi metallo-β-lactamases NDM-1, NDM-5, NDM-6 and NDM-7 in multidrug-resistant Enterobacteriaceae from India

The growing prevalence of carbapenem resistance in Enterobacteriaceae worldwide is a major concern. New Delhi metallo-β-lactamase (NDM)-mediated carbapenem resistance has been identified in Enterobacteriaceae from numerous countries including those of the Indian subcontinent. Currently, seven NDM β-lactamase variants (NDM-1 to -7) have been identified. This study evaluated the detection and molecular characterisation of NDM variants in Enterobacteriaceae at a tertiary care hospital in India. A total of 464 isolates were tested; 57 (12.3%) were resistant or showed reduced susceptibility to imipenem and meropenem. All carbapenem-resistant isolates were blaNDM-positive by PCR, but 13 isolates bore variants that differed in sequence from blaNDM-1. NDM-5, NDM-6 and NDM-7 were identified in two, eight and three isolates, respectively. blaNDM variants were located on plasmids of >100kb with IncF, IncA/C and untypeable replicon types. Genes encoding the 16S rRNA methyltransferases RmtB, RmtC and ArmA as well as those for AmpC β-lactamases were also located on the same plasmids as blaNDM in different combinations. The prevalence of NDM-5 to -7 variants was significantly higher in Escherichia coli (P=0.015) and they were more frequently isolated from the urology ward (P=0.037) than NDM-1. The mortality rate was comparable between patients infected with isolates positive for blaNDM-1 and blaNDM variants [25% (11/44) vs. 23% (3/13)]. Expression of blaNDM variants in E. coli using the same promoter showed that NDM-7 conferred higher resistance to imipenem. The diverse genotypic features of blaNDM indicate rapid evolution of NDM resulting from their wide spread in the Indian subcontinent.

An in silico approach for understanding the molecular evolution of clinically important metallo-beta-lactamases

Resistance to carbapenem, considered to be the drug of last resort for treating serious enterobacterial infections, is a growing problem. Metallo-beta-lactamase (MBL) mediated carbapenem resistance is considered to be clinically most important, since no traditional inhibitors work against them. Hence, we have investigated the changes in drug profile, affinity and binding stability of meropenem with clinically important MBLs viz., IMP, VIM and NDM during the course of molecular evolution. Phylogenetic trees were constructed and amino acids positions, presumed to be exposed to positive selection pressure were analyzed. Based on sequence diversity and selection pressure, IMP-1, IMP-8, IMP-9, IMP-21, IMP-27, IMP-20 and IMP-26 among IMP genes; VIM-1, VIM-2, VIM-13, VIM-29, VIM-18 and VIM-7 among VIM genes and NDM-1 had been selected for in silico analysis. Mode of interaction of selected MBL variants with meropenem were analyzed by Autodock4.0 and LIGPLOT analysis. In all the trajectories, we had found an increase in mouth opening/solvent accessible volume/area of the catalytic pocket and decrease in Gibbs’ free energy (ΔG°) for binding with meropenem and Michealis–Menten constant (Km) – indicating an increase in choice of drugs, binding stability and meropenem affinity from primitive to advanced MBL variants, with exceptions of IMP-20, IMP-26, VIM-13 and VIM-18 which might be due to sign epistasis. Intergenic comparison revealed NDM-1 might have greater drug profile and catalytic efficiency than IMP-1 and VIM-2 due to largest pocket opening and least distance between the Zn-I ion and β-lactam oxygen of meropenem.