A 19-year longitudinal study to characterize carbapenem-nonsusceptible Acinetobacter isolated from patients with bloodstream infections and the contribution of conjugative plasmids to carbapenem resistance and virulence.

BACKGROUND: This study aimed to characterize carbapenem-nonsusceptible Acinetobacter (CNSA) isolated from patients with bacteremia from 1997 to 2015. METHODS: A total of 173 CNSA (12.3%) was recovered from 1403 Acinetobacter isolates. The presence of selected ß-lactamase genes in CNSA was determined by PCR amplification. The conjugation test was used to determine the transferability of metallo-ß-lactamase (MBL)-carrying plasmids. Whole genome sequencing in combination with phenotypic assays was carried out to characterize MBL-plasmids. RESULTS: In general, a trend of increasing numbers of CNSA was observed. Among the 173 CNSA, A. baumannii (54.9%) was the most common species, followed by A. nosocomialis (23.1%) and A. soli (12.1%). A total of 49 (28.3%) CNSA were extensively drug-resistant, and all were A. baumannii. The most common class D carbapenemase gene in 173 CNSA was blaOXA-24-like (32.4%), followed by ISAba1-blaOXA-51-like (20.8%), ISAba1-blaOXA-23 (20.2%), and IS1006/IS1008-blaOXA-58 (11.6%). MBL genes, blaVIM-11,blaIMP-1, and blaIMP-19 were detected in 9 (5.2%), 20 (11.6%), and 1 (0.6%) CNSA isolates, respectively. Transfer of MBL genes to AB218 and AN254 recipient cells was successful for 7 and 6 of the 30 MBL-plasmids, respectively. The seven AB218-derived transconjugants carrying MBL-plasmids produced less biofilm but showed higher virulence to larvae than recipient AB218. CONCLUSIONS: Our 19-year longitudinal study revealed a stable increase in CNSA during 2005-2015. blaOXA-24-like, ISAba1-blaOXA-51-like, and ISAba1-blaOXA-23 were the major determinants of Acinetobacter carbapenem resistance. MBL-carrying plasmids contribute not only to the carbapenem resistance but also to A. baumannii virulence.

Molecular study of blaVIM and blaIMP genes in Acinetobacter baumannii strains isolated from burn patients in Duhok City, Iraq.

INTRODUCTION: Acinetobacter baumannii (A. baumannii) is an opportunistic pathogenic bacterium mainly associated with hospital acquired infections and in immunocompromised individuals who stay in hospitals for a long time. In recent years, it has become increasingly resistant to many different types of antibiotics. The production of the metallo-beta-lactamase (MBL) enzyme is one of the primary causes of this resistance. This study aimed to detect the presence of MBL genes that belong to the verona integrin metallo-ß-lactamase (bla-VIM) and imipenemase (bla-IMP) groups in the isolates of Acinetobacter baumannii from burn patients. METHODOLOGY: One hundred and seventeen (117) isolates of A. baumannii were obtained from patient specimens using traditional methods followed by using the VITEK 2 (BioMérieux, Les Pennes-Mirabeau, France) identification system. Metallo ß-lactamases were detected in the imipenem-resistant strains by using imipenem disks on Muller-Hinton agar. The polymerase chain reaction (PCR) technique was utilized to examine 117 isolates for the detection of MBLs encoding genes such as bla-VIM, and bla-IMP. RESULTS: Imipenem resistance was detected in 78.6% of the patients. The PCR assays of the isolates identified bla-VIM-1, bla-VIM-2, bla-IMP-1 and bla-IMP-2 genes at the rates of 17%, 40.1%, 29.9% and 4.2%, respectively. CONCLUSIONS: The findings suggest that the majority of A. baumannii isolates harbour one or more of the detected genes, signifying that the production of MBLs plays a pivotal role in resistance mechanisms.

Characterization of Verona Integron-Encoded Metallo-ß-Lactamase-Type Carbapenemase-Producing Escherichia coli Isolates Collected over a 16-Year Period in Bolzano (Northern Italy).

Multidrug-resistant Escherichia coli, particularly carbapenemase producers, are a major source of concern. This study aims to investigate the long-term epidemiology of Verona integron-encoded metallo-ß-lactamase (VIM)-producing E. coli in the health district of Bolzano, Northern Italy, by examining the phenotypic and genotypic characteristics of 26 isolates obtained during 2005-2020. Isolates were identified with matrix-assisted laser desorption/ionization time-of-flight, susceptibility testing was by Vitek 2, Sensititre, and Etest; carbapenemase activity was confirmed by Etest and Carbapenemase Inactivation Method (CIM) test; and the VIM-antigen was identified by the NG-Test CARBA 5. Genome sequencing was performed on an Illumina MiSeq platform. Carbapenem minimum inhibitory concentrations varied across methodologies, and overall category agreement between phenotypic methods was low. All 23 sequenced isolates contained blaVIM-1. Eleven (47.8%) isolates belonged to the clonal lineage ST131, with fimH30 being the most common subclone. In Bolzano ST131-fimH30 was present as early as 2005. While the ST131 clonal lineage predominated for the first 10 years, various clonal lineages were present, especially in subsequent years, indicating the concurrent circulation of multiple clonal lineages. Future efforts should focus on the implementation of surveillance methods, including genomic analysis, as well as the use of updated infection control strategies and antibiotic stewardship programs to prevent the spread of these carbapenem-resistant strains.

Coexistence of tmexCD3-toprJ1b tigecycline resistance genes with two novel bla VIM-2-carrying and bla OXA-10-carrying transposons in a Pseudomononas asiatica plasmid.

Introduction: Tigecycline and carbapenems are considered the last line of defense against microbial infections. The co-occurrence of resistance genes conferring resistance to both tigecycline and carbapenems in Pseudomononas asiatica was not investigated. Methods: P. asiatica A28 was isolated from hospital sewage. Antibiotic susceptibility testing showed resistance to carbapenem and tigecycline. WGS was performed to analyze the antimicrobial resistance genes and genetic characteristics. Plasmid transfer by conjugation was investigated. Plasmid fitness costs were evaluated in Pseudomonas aeruginosa transconjugants including a Galleria mellonella infection model. Results: Meropenem and tigecycline resistant P. asiatica A28 carries a 199, 972 bp long plasmid PLA28.4 which harbors seven resistance genes. Sequence analysis showed that the 7113 bp transposon Tn7389 is made up of a class I integron without a 5'CS terminal and a complete tni module flanked by a pair of 25bp insertion repeats. Additionally, the Tn7493 transposon, 20.24 kp long, with a complete 38-bp Tn1403 IR and an incomplete 30-bp Tn1403 IR, is made up of partial skeleton of Tn1403, a class I integron harboring bla OXA-10, and a Tn5563a transposon. Moreover, one tnfxB3-tmexC3.2-tmexD3b-toprJ1b cluster was found in the plasmid and another one in the the chromosome. Furthermore, plasmid PLA28.4 could be conjugated to P. aeruginosa PAO1, with high fitness cost. Discussion: A multidrug-resistant plasmid carrying tmexCD3-toprJ1b and two novel transposons carrying bla VIM-2 and bla OXA-10 -resistant genes was found in hospital sewage, increasing the risk of transmission of antibiotic-resistant genes. These finding highlight the necessary of controlling the development and spread of medication resistance requires continuous monitoring and management of resistant microorganisms in hospital sewage.

Prevalence of Carbapenem-Resistant Enterobacteriaceae in Western Saudi Arabia and Increasing Trends in the Antimicrobial Resistance of Enterobacteriaceae.

PURPOSE:  The aim of the study is to estimate the prevalence rate of carbapenem-resistant Enterobacteriaceae (CRE) and to determine the types of carbapenemase genes present in patients admitted to King Abdulaziz Medical City (KAMC-J) and King Abdulaziz University Hospital (KAUH), both in Jeddah, Saudi Arabia. METHODS:  A total of 180 isolates were analyzed which were included on the basis of retrospective chart review of patients from KAMC-J and KAUH between 1st April 2017 to 30th March 2019. The prevalence of carbapenemase genes ( blaIMP, blaVIM, blaKPC, blaNDM-1, and blaOXA-48) was evaluated by Xpert® Carba-R (Cepheid, Sunnyvale, CA, USA). We assessed the CRE prevalence and described their susceptibility to antimicrobial agents based on antibiogram reports.  Results: Klebsiella pneumoniae showed a higher frequency of bla OXA-48 (79%) than bla NDM (11.7%) genes (p=0.007). The CRE prevalence in KAUH was 8% in 2017 and increased to 13% in 2018. In KAMC-J, the prevalence was 57% in 2018 and 61% in 2019. K. pneumoniae was found to be the most frequently isolated causative organism followed by Escherichia coli . The  bla OXA-48 (76.1%) gene was predominant among overall isolates followed by bla NDM (13.9%); both genes coexisted in 6.1% of the isolates. CONCLUSION:  During the study period, the prevalence of CRE considerably rose in the two tertiary care institutions from western Saudi Arabia. In the CRE isolates, bla OXA-48 was discovered to be the most common gene. We recommend an antimicrobial resistance surveillance system to detect the emergence of resistant genes through use of new rapid diagnostic tests and monitor antimicrobial use in order to improve clinical outcomes of CRE infections given the severity of infection associated with the CRE isolates as well as the limited treatment options available.

Novel Resistance Regions Carrying TnaphA6, blaVIM-2, and blaPER-1, Embedded in an ISPa40-Derived Transposon from Two Multi-Resistant Pseudomonas aeruginosa Clinical Isolates.

Antibiotic resistance is an alarming problem throughout the world and carbapenem-resistant Pseudomonas aeruginosa has been cataloged as critical in the World Health Organization list of microorganisms in urgent need for the development of new antimicrobials. In this work, we describe two novel resistance regions responsible for conferring a multidrug resistance phenotype to two clinical isolates of P. aeruginosa (Pa873 and Pa6415) obtained from patients hospitalized in the ICU of University Hospital of Uruguay. Bacterial identification and antibiotic susceptibility tests were performed using MALDI-TOF and the Vitek 2 system, respectively. WGS was performed for both isolates using Oxford Nanopore Technologies and Illumina and processed by means of hybrid assembly. Both isolates were resistant to ceftazidime, cefepime, piperacillin-tazobactam, aztreonam, and imipenem. Strain Pa6415 also showed resistance to ciprofloxacin. Both strains displayed MICs below the susceptibility breakpoint for CAZ-AVI plus 4 mg/L of aztreonam as well as cefiderocol. Both resistance regions are flanked by the left and right inverted repeats of ISPa40 in two small regions spanning 39.3 and 35.6 kb, for Pa6415 and Pa873, respectively. The resistance region of Pa6415 includes TnaphA6, and the new Tn7516 consists of IRi, In899, qacEΔ1-sul1-ISCR1, qnrVC6-ISCR1-blaPER-1-qacEΔ1-sul1, araJ-like, IS481-like tnpA, ISPa17, and IRR. On the other hand, the resistance region of Pa873 includes Tnaph6 and the new Tn7517 (IRi, In899, qacEΔ1-sul1, ISCR1-blaPER-1-qacEΔ1-sul1, araJ-like, IS481-like tnpA, ISPa17, and IRR). It is necessary to monitor the emergence of genetic structures that threaten to invalidate the available therapeutic resources.

Genotypic Characterization of Carbapenem-Resistant Klebsiella pneumoniae Isolated from an Egyptian University Hospital.

Globally, Klebsiella pneumoniae (K. pneumoniae) has been identified as a serious source of infections. The objectives of our study were to investigate the prevalence of multidrug-resistant (MDR) K. pneumoniae in Tanta University Hospitals, Gharbia Governorate, Egypt; characterize their carbapenem resistance profiles; and identify their different capsular serotypes. We identified and isolated 160 (32%) K. pneumoniae from 500 different clinical samples, performed antimicrobial susceptibility testing, and then used multiplex PCR to detect carbapenemase genes and capsular serotypes K1, K2, K3, K5, K20, K54, and K57. We detected phenotypic carbapenem resistance in 31.3% (50/160) of the isolates; however, molecular assays revealed that 38.75% (62/160) of isolates were carrying carbapenemase-encoding genes. Generally, blaOXA-48 was the prevalent gene (15.5%), followed by blaVIM (15%), blaIMP (7.5%), blaKPC (4%), and blaNDM (3.8%). BlaVIM and blaOXA-48 correlated with phenotypic resistance in 91.67% and 88% of the isolates that harbored them, respectively. Capsular typing showed that the most prevalent pathotype was K1 (30.6%), followed by K57 (24.2%), K54 (19.35%), K20 (9.67%), and K2 (6.45%). A critical risk to community health is posed by the high incidence of multidrug-resistant (MDR) virulent K. pneumoniae isolates from our hospital, and our study examines this pathogen's public health and epidemiological risks.

Plasmids Harboring a Tandem Duplicate of blaVIM-24 in Carbapenem-Resistant ST1816 Pseudomonas aeruginosa in Japan.

The aim of this study was to clarify the biological and clinical significance of a tandem duplicate of blaVIM-24 in Pseudomonas aeruginosa ST1816 isolates. Thirteen ST1816 isolates carrying a plasmid harboring blaVIMs were obtained from two medical settings in Japan between 2016 and 2019. Complete sequencing revealed that, of the 13 plasmids, four had a tandem duplicate of blaVIM-24. These four plasmids harbored a replicon, a relaxase gene, and T4SS genes belonging to IncP-9, MOBF, and MPFT, respectively. All four plasmids transferred to PAO1 by filter mating. Cefepime marginally affected the growth of PAO1, carrying a pUCP19 harboring the tandem duplicate. Western blotting analysis showed that the relative intensity of VIM-24 metallo-ß-lactamase produced by a PAO1 transformant containing a tandem duplicate was 2.6-fold higher than that produced by a PAO1 transformant containing a single copy. These results suggest that the tandem duplicate of blaVIM-24 in plasmids may confer resistance against cefepime, enabling P. aeruginosa ST1816 strains to proliferate in hospitals in Japan.

Phenotypic and genotypic investigation of metallo-ß-lactamases in Pseudomonas aeruginosa clinical isolates in Bushehr, Iran.

Objectives: Production of metallo-ß-lactamases (MBLs) is an important mechanism of resistance to carbapenems. This study aimed to detect the MBL-producing Pseudomonas aeruginosa clinical isolates and to investigate the presence of bla VIM, bla IMP, bla SPM, bla NDM, bla GIM, bla AIM, and bla SIM genes in these isolates in Bushehr, Iran. Materials and Methods: A total of 169 P. aeruginosa clinical isolates were collected from three hospitals in Bushehr. The modified carbapenem inactivation method (mCIM) was used for the phenotypic detection of carbapenemase production. A combination disk test (CDT) was performed for the phenotypic detection of MBL production. To investigate the presence of bla VIM, bla IMP, bla SPM, bla NDM, bla GIM, bla AIM, and bla SIM genes, PCR and sequencing was carried out. Results: Based on the results of mCIM, 40 (23.7%) of 169 isolates were carbapenemase producers. CDT revealed that 26 (15.4%) isolates were MBL producers. bla IMP, bla NDM, and bla VIM genes were found in 18 (69.2%), 8 (30.8%), and 1 (3.8%) of the MBL-producing isolates, respectively. Coexistence of bla IMP and bla NDM was observed in 2 (7.7%) MBL-producing isolates. Among all 169 P. aeruginosa isolates, 23 (13.6%) harbored bla NDM, 18 (10.6%) carried bla IMP, and 1 (0.6%) carried the bla VIM gene. bla SPM, bla GIM, bla AIM, and bla SIM were not found in the present study. Conclusion: bla NDM, bla IMP, and bla VIM genes were detected in this study, which could be a warning sign about the prevalence of these genes among P. aeruginosa clinical isolates in our region. Proper monitoring and detection of MBL-producing isolates are essential steps to prevent the spread of these isolates.

The Molecular Detection of Class B and Class D Carbapenemases in Clinical Strains of Acinetobacter calcoaceticus-baumannii Complex: The High Burden of Antibiotic Resistance and the Co-Existence of Carbapenemase Genes.

The emergence of carbapenem-resistant Acinetobacter calcoaceticus-baumannii complex (CRACB) in clinical environments is a significant global concern. These critical pathogens have shown resistance to a broad spectrum of antibacterial drugs, including carbapenems, mostly due to the acquisition of various ß-lactamase genes. Clinical samples (n = 1985) were collected aseptically from multiple sources and grown on blood and MacConkey agar. Isolates and antimicrobial susceptibility were confirmed with the VITEK-2 system. The modified Hodge test confirmed the CRACB phenotype, and specific PCR primers were used for the molecular identification of blaOXA and blaNDM genes. Of the 1985 samples, 1250 (62.9%) were culture-positive and 200 (43.9%) were CRACB isolates. Of these isolates, 35.4% were recovered from pus samples and 23.5% from tracheal secretions obtained from patients in intensive care units (49.3%) and medical wards (20.2%). An antibiogram indicated that 100% of the CRACB isolates were resistant to ß-lactam antibiotics and ß-lactam inhibitors, 86.5% to ciprofloxacin, and 83.5% to amikacin, while the most effective antibiotics were tigecycline and colistin. The CRACB isolates displayed resistance to eight different AWaRe classes of antibiotics. All isolates exhibited the blaOXA-51 gene, while blaOXA-23 was present in 94.5%, blaVIM in 37%, and blaNDM in 14% of the isolates. The blaOXA-51, blaOXA-23, and blaOXA-24 genes co-existed in 13 (6.5%) isolates. CRACB isolates with co-existing blaOXA-23, blaOXA-24, blaNDM, blaOXA-51 and blaVIM genes were highly prevalent in clinical samples from Pakistan. CRACB strains were highly critical pathogens and presented resistance to virtually all antibacterial drugs, except tigecycline and colistin.

Analysis of carbapenemases genes of carbapenem-resistant Klebsiella pneumoniae isolated from Tehran heart center.

Background and Objectives: Emerging of carbapenem-resistant Klebsiella pneumoniae (CRKP) is one of the major concerns among healthcare systems. This study aimed to investigate the antibiotic susceptibility pattern and carbapenemase genes of carbapenemase-producing K. pneumoniae isolates obtained from Iranian hospitalized patients. Materials and Methods: This study was performed on 71 CRKP strains isolated from different clinical specimens collected in Tehran Heart Center (Tehran, Iran). A Modified Hodge test (MHT) was done for the detection of carbapenemase-producing K. pneumoniae. The presence of bla KPC, bla VIM, bla IMP, bla NDM, and bla OXA-48 -type carbapenemases was evaluated by the PCR method. Results: We identified 8.82% (71/805) of K. pneumoniae isolates as CRKP by MHT test. The antibiotic susceptibility indicated that all isolates were resistant to imipenem, meropenem, cefotaxime, ceftazidime, cefepime, ceftriaxone, cephalothin, ciprofloxacin, and augmentin, and then mostly resistant to aztreonam, cefoxitin, gentamicin, and trimethoprim/sulfamethoxazole with 98.6%, 98.6%, 97.2%, and 94.4%, respectively. The lowest resistance was related to amikacin with 46.5% (33/71 isolates). The level of imipenem MIC for all carbapenem-resistant isolates was estimated ≥32 µg/mL. Among positive isolates for carbapenemase genes, the most frequent gene was bla OXA-48. It was found in 48 (67.6%) isolates followed by bla VIM in 28 (39.4%) isolates. bla IMP, bla NDM, and bla KPC genes were identified in 19 (26.8%), 13 (18.3%) and 5 (7.0%) isolates, respectively. These genes were not detected in nine isolates. Conclusion: The relatively high frequency of some carbapenemase genes suggests major concern about the emergence of isolates containing carbapenem resistance genes as a potential health threat.

Impact of Peptidoglycan Recycling Blockade and Expression of Horizontally Acquired ß-Lactamases on Pseudomonas aeruginosa Virulence.

In the current scenario of antibiotic resistance magnification, new weapons against top nosocomial pathogens like Pseudomonas aeruginosa are urgently needed. The interplay between ß-lactam resistance and virulence is considered a promising source of targets to be attacked by antivirulence therapies, and in this regard, we previously showed that a peptidoglycan recycling blockade dramatically attenuated the pathogenic power of P. aeruginosa strains hyperproducing the chromosomal ß-lactamase AmpC. Here, we sought to ascertain whether this observation could be applicable to other ß-lactamases. To do so, P. aeruginosa wild-type or peptidoglycan recycling-defective strains (ΔampG and ΔnagZ) harboring different cloned ß-lactamases (transferable GES, VIM, and OXA types) were used to assess their virulence in Galleria mellonella larvae by determining 50% lethal doses (LD50s). A mild yet significant LD50 increase was observed after peptidoglycan recycling disruption per se, whereas the expression of class A and B enzymes did not impact virulence. While the production of the narrow-spectrum class D OXA-2 entailed a slight attenuation, its extended-spectrum derivatives OXA-226 (W159R [bearing a change of W to R at position 159]), OXA-161 (N148D), and principally, OXA-539 (D149 duplication) were associated with outstanding virulence impairments, especially in recycling-defective backgrounds (with some LD50s being >1,000-fold that of the wild type). Although their exact molecular bases remain to be deciphered, these results suggest that mutations affecting the catalytic center and, therefore, the hydrolytic spectrum of OXA-2-derived enzymes also drastically impact the pathogenic power of P. aeruginosa. This work provides new and relevant knowledge to the complex topic of the interplay between the production of ß-lactamases and virulence that could be useful to build future therapeutic strategies against P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is one of the leading nosocomial pathogens whose growing resistance makes the development of therapeutic options extremely urgent. The resistance-virulence interplay has classically aroused researchers' interest as a source of therapeutic targets. In this regard, we describe a wide array of virulence attenuations associated with different transferable ß-lactamases, among which the production of OXA-2-derived extended-spectrum ß-lactamases stood out as a dramatic handicap for pathogenesis, likely as a side effect of mutations causing the expansion of their hydrolytic spectrums. Moreover, our results confirm the validity of disturbing peptidoglycan recycling as a weapon to attenuate P. aeruginosa virulence in class C and D ß-lactamase production backgrounds. In the current scenario of dissemination of horizontally acquired ß-lactamases, this work brings out new data on the complex interplay between the production of specific enzymes and virulence attenuation that, if complemented with the characterization of the underlying mechanisms, will likely be exploitable to develop future virulence-targeting antipseudomonal strategies.

Escherichia coli ST471 Producing VIM-4 Metallo-ß-Lactamase in Colombia.

An Escherichia coli isolate sequence-type 471 (ST471) producing Verona integron-encoded metallo-ß-lactamases (VIM)-4 was recovered from a rectal swab in a patient without travel records with osteomyelitis in Colombia. The isolate carried a class 1 integron-borne blaVIM-4 gene with a 170-bp duplication in the 3' end of the gene, preceded by an aac(6')-Ib gene. The genetic environment of blaVIM-4, blaCMY-2, and sul2 genes showed similarities to the backbone of pKKp4, an IncA/C-type plasmid from a Klebsiella pneumoniae strain carrying blaVIM-4 recovered in Kuwait. This is the first report of blaVIM-4 in Enterobacterales in South America. Our results suggest that blaVIM-4 gene was found on an IncA/C-type plasmid that could play a role in the spread of VIM-4 carbapenemase in Colombia.

Association of Biofilm Inducer with bla VIM, bla IMP, and bla NDM in Pseudomonas aeruginosa Isolates.

Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous opportunistic organism that is hard to treat. This study aimed to investigate the association of bla VIM, bla IMP, and bla NDM prevalence with Cyclic di-GMP (c-di-GMP) in P. aeruginosa. To this end, 27 clinical isolates of P. aeruginosa were obtained from different hospitals in Baghdad, Iraq. The phenotypic detection of carbapenem and biofilm assays was performed by the M63 minimal medium, supplemented with glucose, magnesium sulfate. The polymerase chain reaction was utilized to detect carbapenem genes. The results showed that the isolates were highly resistant to Imipenem (37%) and Meropenem (63%). Imipenem (37%) and Meropenem (63%) demonstrated a moderate sensitivity against P. aeruginosa. The P. aeruginosa No.5 showed high resistance to carbapenem by bla VIM +, bla IMP +, and bla NDM +, followed by a robust biofilm confirmed with c-di-GMP levels and the twitching motility ability. Upon these findings, the use of antibiotics should be restricted to severe bacterial infections to avoid the rapid emergence of new resistant isolates, which leads to the hard treatment of infection with P. aeruginosa. It is highly recommended that these findings be notified for infectious control. Future studies can investigate the link between transferable resistant genes and c-di-GMP values.

Dissemination of carbapenem resistant bacteria from hospital wastewater into the environment.

Infections with antibiotic resistant pathogens threaten lives and cause substantial costs. For effective interventions, knowledge of the transmission paths of resistant bacteria to humans is essential. In this study, carbapenem resistant bacteria were isolated from the wastewater of a maximum care hospital during a period of two years, starting in the patient rooms and following the sewer system to the effluent of the wastewater treatment plant (WWTP). The bacteria belonged to six different species and 44 different sequence types (STs). The most frequent STs, ST147 K. pneumoniae (blaNDM/blaOXA-48) and ST235 P. aeruginosa (blaVIM) strains, were present at nearly all sampling sites from the hospital to the WWTP effluent. After core genome multi-locus sequence typing (cgMLST), all ST147 K. pneumoniae strains presented a single epidemiological cluster. In contrast, ST235 P. aeruginosa formed five cgMLST clusters and the largest cluster contained the strain from the WWTP effluent, indicating without doubt, a direct dissemination of both high-risk clones into the environment. Thus, there are - at least two - possible transmission pathways to humans, (i) within the hospital by contact with the drains of the sanitary installations and (ii) by recreational or irrigation use of surface waters that have received WWTP effluent. In conclusion, remediation measures must be installed at both ends of the wastewater system, targeting the drains of the hospital as well as at the effluent of the WWTP.

Implication of different replicons in the spread of the VIM-1-encoding integron, In110, in Enterobacterales from Czech hospitals.

Background: VIM metallo-ß-lactamases are enzymes characterized by the ability to hydrolyze all ß-lactams. Usually, bla VIM-like genes are carried by class 1 integrons. In the Czech Republic, only sporadic cases of VIM-producing Enterobacterales have been reported in which those isolates carried the VIM-1 carbapenemase-encoding integron In110. However, during 2019-2020, an increased number was reported. Therefore, the aim of the current study was to characterize the genetic elements involved in the increased spread of bla VIM genes. Materials and methods: 32 VIM-producing Enterobacterales collected between 2019 and 2020 were subjected to: antimicrobial susceptibility testing, integron analysis, and short reads sequencing. Based on the results, 19 isolates were selected as representative and sequenced using Sequel I platform. Results: The 32 VIM-producing isolates exhibited variations in the MICs of carbapenems. Based on short-read data, 26 of the 32 sequenced isolates harbored the bla VIM-1 allele while six isolates carried the bla VIM-4 gene. The most prevalent was the In110 integron (n = 24) and two isolates carried the In4873 class 1 integron. The bla VIM-4 allele was identified in class 1 integrons In1174 (n = 3), In416 (n = 1), In2143 (n = 1) and In2150. Long reads sequencing revealed that the bla VIM was carried by: pKPC-CAV1193-like (n = 6), HI1 (pNDM-CIT; n = 4), HI2 (n = 3), FIB (pECLA; n = 2) and N (n = 1) incompatibility groups. Two bla VIM-carrying plasmids could not be typed by the database, while another one was integrated into the chromosome. Conclusion: We observed the spread of VIM-encoding integrons, mainly of In110, among Enterobacterales isolated from Czech hospitals, but also an increased number of novel elements underlining the ongoing evolution.

Development of a novel loop-mediated isothermal amplification assay for ß-lactamase gene identification using clinical isolates of Gram-negative bacteria.

Rapid evaluation of antimicrobial susceptibility is important in the treatment of nosocomial infections by Gram-negative bacteria, which increasingly carry carbapenemases and metallo-ß-lactamases. We developed loop-mediated isothermal amplification (LAMP)-based assays for four ß-lactamase genes (bla KPC, bla NDM-1, bla IMP-1 group, and bla VIM). The assays were evaluated using eight reference bacterial strains (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter bereziniae) harboring six ß-lactamase genes. A total of 55 Gram-negative bacterial strains, including 47 clinical P. aeruginosa isolates, fully characterized by next-generation sequencing (NGS), were used to evaluate the LAMP assays. The results were compared to those of conventional PCR. The LAMP assays were able to detect as few as 10 to 100 copies of a gene, compared to 10 to 104 copies for conventional PCR. The LAMP assay detected four ß-lactamase genes with a sensitivity similar to that using purified DNA as the template in DNA-spiked urine, sputum, and blood specimens. By contrast, the sensitivity of PCR was 1- to 100-fold lower with DNA-spiked clinical specimens. Therefore, the LAMP assays were proved to be an appropriate tool for the detection of four ß-lactamases.

Genetic diversity and molecular analysis of metallo beta lactamases among imipenem resistant clinical isolates of Pseudomonas aeruginosa from Peshawar, Pakistan.

OBJECTIVES: Pseudomonas aeruginosa is an opportunistic pathogen with remarkable adaptation ability to thrive in diverse environmental conditions. This study aimed at phenotypic and molecular analysis of metallo beta lactamases (blaIMP, blaVIM, blaNDM-1 and blaSPM-1) and genetic diversity analysis among imipenem resistant clinical isolates of Pseudomonas aeruginosa. METHODS: This study was conducted from May 2017 to June 2018. The study included 187 Pseudomonas aeruginosa isolates collected from different clinical specimens from Peshawar, Pakistan. The isolates were analyzed for resistance to imipenem. Combined disc test (CDT) was then performed for phenotypic detection of metallo beta lactamases among imipenem resistant isolates of Pseudomonas aeruginosa. Molecular detection of metallo beta lactamases genes i.e. blaIMP, blaVIM, blaNDM-1 and blaSPM-1 was analyzed through polymerase chain reaction. Genetic diversity was determined through RAPD-PCR. RESULTS: MBL production was observed in 76% (n=19) isolates. The occurrence of MBL genes blaIMP, blaNDM-1 and blaVIM was 68% (n=17), 48% (n=12), and 4% (n=1) respectively. The blaSPM-1 gene was not detected. High genetic diversity was observed in current study. Out of 182 isolates 171 isolates showed different RAPD profiles (93.95% polymorphism); 160 were unique RAPD strains and based on similarity coefficient ≥ 80%, 22 isolates were clustered into 11 distinct clones. CONCLUSION: A high prevalence of blaIMP and blaNDM-1 among imipenem resistant isolates of Pseudomonas aeruginosa is alarming that calls for proper control and prevention strategies. RAPD technique was found to be a good genotyping technique when limited resources are available.

Genetic Diversity of Multidrug-Resistant Pseudomonas aeruginosa Isolates Carrying bla VIM-2 and bla KPC-2 Genes That Spread on Different Genetic Environment in Colombia.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen with an increase in the frequency of infections caused by multidrug resistant (MDR) and extensively drug resistant (XDR) strains, limiting the available therapeutic options. The most troublesome resistance is the acquisition and production of carbapenemases such as Verona integron-encoded metallo-ß-lactamases (VIM), the most frequent and widespread, and the Klebsiella pneumoniae carbapenemases (KPC), which has continuously spread in the last decade. Its dissemination is linked to their location on mobile genetic elements (MGEs). In Colombia, VIM and KPC have been increasing in its frequency showing major successful dissemination. In this article, we molecularly characterized and analyzed the genetic context of bla VIM and bla KPC in carbapenem-resistant P. aeruginosa (CRPA) isolates from infected and colonized patients in two tertiary-care hospitals, one in Medellín and the other in a municipality close to Medellín, both areas with high carbapenemase endemicity in Colombia (2013-2015). Using whole-genome sequencing (WGS), we identified a remarkable variety of genetic backgrounds in these MDR P. aeruginosa isolates carrying bla KPC- 2 and bla VIM- 2. There were a diversity of class 1 integron and variations in the gene cassettes associated to bla VIM- 2, as well as a possible event of spread of bla KPC- 2 mediated by a plasmid that contained part of Tn4401b in one infection case. The dissemination of bla VIM- 2 and bla KPC- 2 in P. aeruginosa in this area in Colombia has been strongly influenced by successful international clones, carrying these genes and additional determinants of resistance on MGEs, accompanied by gene rearrangement under an antimicrobial selection pressure. These findings emphasize the need to implement control strategies based on rational antibiotic use.

Emergence and Genetic Characterization of Plasmid-Encoded VIM-2-Producing Pseudomonas stutzeri with Novel Integron In1998 Isolated from Cerebrospinal Fluid.

PURPOSE: To investigate the genomic and plasmid characteristics of a newly discovered Pseudomonas stutzeri strain with a bla VIM-2-carrying plasmid and novel integron In1998 isolated from a cerebrospinal fluid specimen in a teaching hospital. METHODS: Species identification was performed by MALDI-TOF MS, and bla VIM-2 was identified by PCR and Sanger sequencing. Whole-genome sequencing analysis was conducted using the Illumina NovaSeq 6000 and Oxford Nanopore platforms. Integron detection was performed using INTEGRALL. The phylogenetic tree was constructed by using kSNP3.0. Plasmid characteristics were assessed by S1-pulsed-field gel electrophoresis (S1-PFGE), Southern blotting, conjugation experiments, and whole-genome sequencing analysis. Comparative genomics analysis of the plasmid and genetic context of bla VIM-2 were conducted by using BLAST Ring Image Generator (BRIG) and Easyfig 2.3, respectively. RESULTS: ZDHY95, an MDR strain of P. stutzeri harboring bla VIM-2, was identified. It was sensitive only to amikacin and was resistant to carbapenems, ß-lactams, aztreonam, fluoroquinolones, and aminoglycosides. Joint S1-PFGE, Southern blot, conjugation assay, and whole-genome sequencing experiments confirmed that the bla VIM-2 gene was located within class I integron In1722 of the plasmid and that the surrounding genetic environment was 5'CS-aacA4'-30-bla VIM-2-aacA4'-3'CS. The novel class I integron In1998 was detected on the chromosome of P. stutzeri ZDHY95, and the gene cassette array was 5'CS-aacA3-aadA13-cmlA8-bla OXA-246-arr3-dfrA27-3'CS. Phylogenetic analysis showed that antimicrobial resistance gene-carrying P. stutzeri isolates were divided into two clusters, mainly containing isolates from the USA and Pakistan. CONCLUSION: A novel bla VIM-2-carrying conjugative plasmid, pZDHY95-VIM-2, was reported for the first time in P. stutzeri, elucidating the genetic environment and transfer mechanism. The gene structure of the novel class I integron In1998 was also clarified. We explored the phylogenetic relationship of P. stutzeri with drug resistance genes and suggested that Pseudomonas with metallo-ß-lactamases (MBLs) in the hospital environment may cause infection in patients with long-term intubation or after interventional surgery.