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OXA-244, an R214G variant of OXA-48, is silently spreading worldwide likely because of difficulties in detection using classical screening media. Here, we characterized two clinical isolates of Escherichia coli and Citrobacter youngae that displayed reduced susceptibility to carbapenems but were lacking significant carbapenemase activity as revealed by negative Carba NP test results. However, positive test results were seen for OXA-48-like enzymes by lateral flow immunoassays. WGS revealed the presence of a blaOXA-181-like gene that codes for OXA-484, an R214G variant of OXA-181. BlaOXA-484 gene was located on a 58.4-kb IncP1-like plasmid (pN-OXA-484), that upon transfer into E. coli HB4 with impaired permeability, conferred carbapenem and temocillin resistance (MICs > 32 mg/L). E. coli TOP10 (pTOPO-OXA-484) revealed reduced MICs in most substrates as compared to E. coli TOP10 (pTOPO-OXA-181), especially for imipenem (0.25 mg/L versus 0.75 mg/L) and temocillin (16 mg/L versus 1028 mg/L). Catalytic efficiencies of OXA-484 were reduced as compared to OXA-181 for most ß-lactams including imipenem and temocillin with 27.5- and 21.7-fold reduction, respectively. Molecular modeling confirmed that the salt bridges between R214, D159, and the R1 substituent's carboxylate group of temocillin were not possible with G214 in OXA-484, explaining the reduced affinity for temocillin. In addition, changes in active site's water network may explain the decrease in hydrolysis rate of carbapenems. OXA-484 has weak imipenem and temocillin hydrolytic activities, which may lead to silent spread due to underdetection using selective screening media or biochemical imipenem hydrolysis confirmatory tests. [ABSTRACT FROM AUTHOR]

Waste Water Treatment Plants (WWTP) aim to reduce contamination in effluent water; however, studies indicate antimicrobial resistance genes (ARGs) persist post-treatment, potentially leading to their spread from human populated areas into the environment. This study evaluated the impact of a large WWTP serving 125,000 people on the Iskar River in Bulgaria, by characterizing the spatial and short-term temporal dynamics in bacterial community dynamics and resistance profiles of the surface water. Pairs of samples were collected biweekly on four dates from two different locations, one about 800 m after the WWTP effluents and the other 10 km downstream. Taxonomic classification revealed the dominance of Pseudomonodota and Bacteriodota, notably the genera Flavobacterium, Aquirufa, Acidovorax, Polynucleobacter, and Limnohabitans. The taxonomic structure corresponded with both lentic and lotic freshwater habitats, with Flavobacterium exhibiting a significant decrease over the study period. Principal Coordinate Analysis revealed statistically significant differences in bacterial community composition between samples collected on different dates. Differential abundance analysis identified notable enrichment of Polynucleobacter and Limnohabitans. There were shifts within the enriched or depleted bacterial taxa between early and late sampling dates. High relative abundance of the genes erm(B), erm(F), mph(E), msr(E) (macrolides); tet(C), tet(O), tet(W), tet(Q) and tet(X) (tetracyclines); sul1 and sul2 (sulphonamides); and cfxA3, cfxA6 (beta-lactams) were detected, with trends of increased presence in the latest sampling dates and in the location closer to the WWTP. Of note, genes conferring resistance to carbapenems blaOXA-58 and blaIMP-33-like were identified. Co-occurrence analysis of ARGs and mobile genetic elements on putative plasmids showed few instances, and the estimated human health risk score (0.19) according to MetaCompare2.0 was low. In total, 29 metagenome-assembled genomes were recovered, with only a few harbouring ARGs. This study enhances our understanding of freshwater microbial community dynamics and antibiotic resistance profiles, highlighting the need for continued ARGs monitoring. [ABSTRACT FROM AUTHOR]

Multidrug-resistant (MDR) bacteria have become one of the most important health problems. We aimed to assess whether international travel may facilitate their spread through the colonization of asymptomatic travelers. A cross-sectional study was conducted (November 2018 to February 2022). Pharyngeal and rectal swabs were obtained from long-term travelers and recently arrived migrants from non-European countries, and an epidemiological survey was performed. Colonization by Gram-negative bacteria and methicillin-resistant Staphylococcus aureus (MRSA) was determined by chromogenic media and MALDI-TOF-MS. Resistance mechanisms were determined by the biochip-based molecular biology technique. Risk factors for colonization were assessed by logistic regression. In total, 122 participants were included: 59 (48.4%) recently arrived migrants and 63 (51.6%) long-term travelers. After their trip, 14 (11.5%) participants—5 (8.5%) migrants and 9 (14.3%) travelers—had rectal colonization by one MDR bacterium. Escherichia coli carrying the extended-spectrum beta-lactamase (ESBL) CTX-M-15 was the most frequent. No participants were colonized by MRSA or carbapenemase-producing Enterobacteriaceae. The only risk factor independently associated with MDR bacterial colonization was previous hospital attention [OR, 95% CI: 10.16 (2.06–50.06)]. The risk of colonization by MDR bacteria among recently arrived migrants and long-term travelers is similar in both groups and independently associated with previous hospital attention. [ABSTRACT FROM AUTHOR]

Carbapenems are last-resort antibiotics used to treat multidrug-resistant bacterial infections. Resistance to carbapenems has been designated as an urgent threat and is increasing in healthcare settings. However, little is still known about the distribution and characteristics of carbapenem-resistant bacteria (CRB) outside of healthcare settings. Here, we surveyed the distribution of CRB in ten diverse freshwater and seawater environments in California, U.S., ranging from San Luis Obispo County to San Bernardino County, combining both direct isolation and enrichment approaches to increase the diversity of isolated CRB. From the locations surveyed, we selected 30 CRB for further characterization. These isolates were identified as members of the genera Aeromonas, Enterobacter, Enterococcus, Paenibacillus, Pseudomonas, Sphingobacterium, and Stenotrophomonas. These isolates were resistant to carbapenems, other β-lactams, and often to other antibiotics (tetracycline, gentamicin, or ciprofloxacin). We also found that nine isolates belonging to the genera Aeromonas, Enterobacter (blaIMI-2), and Stenotrophomonas (blaL1) produced carbapenemases. Overall, our findings indicate that sampling different types of aquatic environments and combining different isolation approaches increase the diversity of the environmental CRB obtained. Moreover, our study supports the increasingly recognized role of natural water systems as an underappreciated reservoir of bacteria resistant to carbapenems and other antibiotics, including bacteria carrying carbapenemase genes. [ABSTRACT FROM AUTHOR]

Antibiotic resistance remains one of the most pressing public health issues facing the world today. At the forefront of this battle lies the ever-increasing identification of extended-spectrum beta-lactamases and carbapenemases within human pathogens, conferring resistance towards broad-spectrum and last-resort antimicrobials. This study was prompted due to the identification of a pathogenic Aeromonas hydrophila isolate (strain MAH-4) collected from abdominal fluid, which presented a robust resistance pattern against second-, third-, and fourth-generation cephalosporins, ertapenem, ciprofloxacin, gentamicin, levofloxacin and moxifloxacin, and beta lactam/beta-lactamase inhibitor combinations. Whole genome sequencing was performed and identified a 328 kb plasmid (pMAH4) encoding 10 antibiotic resistance genes, including blaSFO-1, blaTEM-1, and blaOXA-1 of A. hydrophia MAH-4. This is the first report of beta-lactamase SFO-1 within a clinical strain of Aeromonas. Due to the remarkable sequence identity of pMAH4 to plasmids associated with Enterobacterales genera like Klebsiella and the extensive capabilities of Aeromonas for horizontal gene transfer, our identification of a clinical isolate encoding SFO-1 on a plasmid suggests antibiotic resistance gene mobility between Enterobacterales and non-Enterobacterales species. [ABSTRACT FROM AUTHOR]

Background: The emergence and global spread of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) are of great concern to health services worldwide. These β-lactamases hydrolyze almost all β-lactams, are plasmid-encoded, and are easily transferable among bacterial species. They are mostly of the KPC types in CR-hvKp. OXA-48-producing hvKP strains have been rarely reported in the literature. Methods: OXA-48-producing hvKP strains were collected from clinical specimens at the First Affiliated Hospital of Hainan Medical University from January 2022 to March 2023. Hypervirulent strains were tested for virulence in a mouse lethality study and underwent whole genome sequencing to identify genomic features. Results: A total of 42 unique OXA-48-bearing K. pneumoniae strains were identified, including three CR-hvKP strains (KP2683-1, NCRE61, and KP2185), which were isolated from bacteremia, pulmonary abscess, and liver abscess separately. The three CR-hvKP strains belonged to two different clones of ST11 KL64 (KP2185 and NCRE61) and ST23 K1 (KP2683-1). The KP2683-1 strain had the highest virulence. Whole genome sequencing analysis indicated that NCRE61 and KP2185 acquired IncFIB-type plasmids with a set of virulence genes (iroBCDN, iucABCD, iutA, rmpA, and rmpA2), while KP2683-1 acquired an IncL-type blaOXA-48-harboring plasmid. Consecutive cultures showed that the blaOXA-48-harboring plasmids were highly stable in the three hvKP strains and could be transmitted to Escherichia coli J53 by conjugation. The drug susceptibility testing results show that Ceftazidime/avibactam is sensitive for OXA-48-producing hvKP. Conclusions: Our study highlighted the two evolutionary pathways of OXA-48-producing hvKP strains and confirmed their virulence through in vivo testing. Ceftazidime/avibactam may be a viable option for treating OXA-48-producing hvKP strains. [ABSTRACT FROM AUTHOR]

OXA-244, an R214G variant of OXA-48, is silently spreading worldwide likely because of difficulties in detection using classical screening media. Here, we characterized two clinical isolates of Escherichia coli and Citrobacter youngae that displayed reduced susceptibility to carbapenems but were lacking significant carbapenemase activity as revealed by negative Carba NP test results. However, positive test results were seen for OXA-48-like enzymes by lateral flow immunoassays. WGS revealed the presence of a blaOXA-181-like gene that codes for OXA-484, an R214G variant of OXA-181. BlaOXA-484 gene was located on a 58.4-kb IncP1-like plasmid (pN-OXA-484), that upon transfer into E. coli HB4 with impaired permeability, conferred carbapenem and temocillin resistance (MICs > 32 mg/L). E. coli TOP10 (pTOPO-OXA-484) revealed reduced MICs in most substrates as compared to E. coli TOP10 (pTOPO-OXA-181), especially for imipenem (0.25 mg/L versus 0.75 mg/L) and temocillin (16 mg/L versus 1028 mg/L). Catalytic efficiencies of OXA-484 were reduced as compared to OXA-181 for most ß-lactams including imipenem and temocillin with 27.5- and 21.7-fold reduction, respectively. Molecular modeling confirmed that the salt bridges between R214, D159, and the R1 substituent’s carboxylate group of temocillin were not possible with G214 in OXA-484, explaining the reduced affinity for temocillin. In addition, changes in active site’s water network may explain the decrease in hydrolysis rate of carbapenems. OXA-484 has weak imipenem and temocillin hydrolytic activities, which may lead to silent spread due to underdetection using selective screening media or biochemical imipenem hydrolysis confirmatory tests.

Waste Water Treatment Plants (WWTP) aim to reduce contamination in effluent water; however, studies indicate antimicrobial resistance genes (ARGs) persist post-treatment, potentially leading to their spread from human populated areas into the environment. This study evaluated the impact of a large WWTP serving 125,000 people on the Iskar River in Bulgaria, by characterizing the spatial and short-term temporal dynamics in bacterial community dynamics and resistance profiles of the surface water. Pairs of samples were collected biweekly on four dates from two different locations, one about 800 m after the WWTP effluents and the other 10 km downstream. Taxonomic classification revealed the dominance of Pseudomonodota and Bacteriodota, notably the genera Flavobacterium, Aquirufa, Acidovorax, Polynucleobacter, and Limnohabitans. The taxonomic structure corresponded with both lentic and lotic freshwater habitats, with Flavobacterium exhibiting a significant decrease over the study period. Principal Coordinate Analysis revealed statistically significant differences in bacterial community composition between samples collected on different dates. Differential abundance analysis identified notable enrichment of Polynucleobacter and Limnohabitans. There were shifts within the enriched or depleted bacterial taxa between early and late sampling dates. High relative abundance of the genes erm(B), erm(F), mph(E), msr(E) (macrolides); tet(C), tet(O), tet(W), tet(Q) and tet(X) (tetracyclines); sul1 and sul2 (sulphonamides); and cfxA3, cfxA6 (beta-lactams) were detected, with trends of increased presence in the latest sampling dates and in the location closer to the WWTP. Of note, genes conferring resistance to carbapenems blaOXA-58 and blaIMP-33-like were identified. Co-occurrence analysis of ARGs and mobile genetic elements on putative plasmids showed few instances, and the estimated human health risk score (0.19) according to MetaCompare2.0 was low. In total, 29 metagenome-assembled genomes were recovered, with only a few harbouring ARGs. This study enhances our understanding of freshwater microbial community dynamics and antibiotic resistance profiles, highlighting the need for continued ARGs monitoring.

In Kuwait, some sewage is discharged into the sea untreated, causing a health risk. Previously, we investigated the presence of pathogenic E. coli among the 140 isolates of E. coli cultured from the raw sewage from three sites in Kuwait. The aim of the current study was to characterize the antimicrobial resistance of these isolates and the implications of resistance. Susceptibility to 15 antibiotic classes was tested. Selected genes mediating resistance to cephalosporins and carbapenems were sought. ESBL and carbapenemase production were also determined. Two virulent global clones, ST131 and ST648, were sought. A total of 136 (97.1%), 14 (10.0%), 128 (91.4%), and 2 (1.4%) isolates were cephalosporin-resistant, carbapenem-resistant, multidrug-resistant (MDR), and extensively drug-resistant (XDR), respectively. Among the cephalosporin-resistant isolates, ampC, blaTEM, blaCTX-M, blaOXA-1, and blaCMY-2 were found. Eighteen (12.9%) samples were ESBL producers. All carbapenem-resistant isolates were negative for carbapenemase genes (blaOXA-48, blaIMP, blaGES, blaVIM, blaNDM, and blaKPC), and for carbapenemase production. Resistance rates in carbapenem-resistant isolates to many other antibiotics were significantly higher than in susceptible isolates. A total of four ST131 and ST648 isolates were detected. The presence of MDR and XDR E. coli and global clones in sewage poses a threat in treating E. coli infections. [ABSTRACT FROM AUTHOR]

Carbapenems are atypical β-lactam antibiotics with a broade antibacterial spectrum and strong antibacterial activity; however, the emergence and spread of carbapenemases have led to a decline in their effectiveness. New Delhi metallo-β-lactamase (NDM) is an important carbapenemase that has attracted widespread attention and poses a major threat to public health. To investigate the epidemiological characteristics of blaNDM in swine and chicken farms in southwestern China, we isolated 102 blaNDM-positive Enterobacterales strains from 18 farms in Sichuan and Yunnan provinces in 2021, with Escherichia coli and Klebsiella spp. being the main reservoirs of blaNDM, variant blaNDM-5 being the most prevalent, and all strains being multi-drug resistant. Whole-genome sequencing analysis of 102 blaNDM-positive Enterobacterales strains revealed that blaNDM had spread primarily through its carriers on the same farm and among the 18 farms in this study. A high degree of genetic similarity between animal-derived blaNDM-positive Escherichia coli strains and human-derived strains was also identified, suggesting a potential mutual transmission between them. Nanopore sequencing results indicated that blaNDM is predominantly present on the IncX3 plasmid, that an insertion sequence might be important for recombination in the blaNDM genetic environment, and that most of the plasmids carrying blaNDM are transferable. Collectively, our results enrich the current epidemiological information regarding blaNDM in pig and chicken farms in Southwest China, revealing its transmission pattern, as well as the potential risk of transmission to humans, which could help to better understand and control the spread of blaNDM. [ABSTRACT FROM AUTHOR]

(1) Background: The purpose of the study was to describe the activity of mex efflux pumps in Multidrug-Resistant (MDR) clinical isolates of Pseudomonas aeruginosa and to compare the carbapenem-resistance identification tests with PCR; (2) Methods: Sixty MDR P. aeruginosa were analyzed for detection of carbapenemase by disk diffusion inhibitory method, carbapenem inactivation method and Modified Hodge Test. Endpoint PCR was used to detect 7 carbapenemase genes (blaKPC, blaOXA48-like, blaNDM, blaGES-2, blaSPM, blaIMP, blaVIM) and mcr-1 for colistin resistance. The expression of mexA, mexB, mexC, mexE and mexX genes corresponding to the four main efflux pumps was also evaluated; (3) Results: From the tested strains, 71.66% presented at least one carbapenemase gene, with blaGES-2 as the most occurring gene (63.3%). Compared with the PCR, the accuracy of phenotypic tests did not exceed 25% for P. aeruginosa. The efflux pump genes were present in all strains except one. In 85% of the isolates, an overactivity of mexA, mexB and mostly mexC was detected. Previous treatment with ceftriaxone increased the activity of mexC by more than 160 times; (4) Conclusions: In our MDR P. aeruginosa clinical isolates, the carbapenem resistance is not accurately detected by phenotypic tests, due to the overexpression of mex efflux pumps and in a lesser amount, due to carbapenemase production. [ABSTRACT FROM AUTHOR]

The emergence of carbapenem-resistant Enterobacterales (CRE) has been recognized as a significant concern globally. Ceftazidime/avibactam (CZA) is a novel β-lactam/β-lactamase inhibitor that has demonstrated activity against isolates producing class A, C, and D β-lactamases. Here-in, we evaluated the in vitro activity of CZA and comparator antimicrobial agents against 858 CRE isolates, arising from the Southeast Asian region, collected from a large tertiary hospital in Singapore. These CRE isolates mainly comprised Klebsiella pneumoniae (50.5%), Escherichia coli (29.4%), and Enterobacter cloacae complex (17.1%). Susceptibility rates to levofloxacin, imipenem, meropenem, doripenem, aztreonam, piperacillin/tazobactam, cefepime, tigecycline, and polymyxin B were low. CZA was the most active β-lactam agent against 68.9% of the studied isolates, while amikacin was the most active agent among all comparator antibiotics (80% susceptibility). More than half of the studied isolates (51.4%) identified were Klebsiella pneumoniae carbapenemase (KPC)-2 producers, 25.9% were New Delhi metallo-β-lactamase (NDM) producers, and Oxacillinase (OXA)-48-like producers made up 10.7%. CZA was the most active β-lactam agent against KPC-2, OXA-48-like, and Imipenemase (IMI) producers (99.3% susceptible; MIC50/90: ≤1/2 mg/L). CZA had excellent activity against the non-carbapenemase-producing CRE (91.4% susceptible; MIC50/90: ≤1/8 mg/L). Expectedly, CZA had no activity against the metallo-β-lactamases (MBL)-producing CRE (NDM- and Imipenemase MBL (IMP) producers; 27.2% isolates), and the carbapenemase co-producing CRE (NDM + KPC, NDM + OXA-48-like, NDM + IMP; 3.0% isolates). CZA is a promising addition to our limited armamentarium against CRE infections, given the reasonably high susceptibility rates against these CRE isolates. Careful stewardship and rational dosing regimens are required to preserve CZA's utility against CRE infections. [ABSTRACT FROM AUTHOR]

Multidrug-resistant (MDR) bacteria have become one of the most important health problems. We aimed to assess whether international travel may facilitate their spread through the colonization of asymptomatic travelers. A cross-sectional study was conducted (November 2018 to February 2022). Pharyngeal and rectal swabs were obtained from long-term travelers and recently arrived migrants from non-European countries, and an epidemiological survey was performed. Colonization by Gram-negative bacteria and methicillin-resistant Staphylococcus aureus (MRSA) was determined by chromogenic media and MALDI-TOF-MS. Resistance mechanisms were determined by the biochip-based molecular biology technique. Risk factors for colonization were assessed by logistic regression. In total, 122 participants were included: 59 (48.4%) recently arrived migrants and 63 (51.6%) long-term travelers. After their trip, 14 (11.5%) participants—5 (8.5%) migrants and 9 (14.3%) travelers—had rectal colonization by one MDR bacterium. Escherichia coli carrying the extended-spectrum beta-lactamase (ESBL) CTX-M-15 was the most frequent. No participants were colonized by MRSA or carbapenemase-producing Enterobacteriaceae. The only risk factor independently associated with MDR bacterial colonization was previous hospital attention [OR, 95% CI: 10.16 (2.06–50.06)]. The risk of colonization by MDR bacteria among recently arrived migrants and long-term travelers is similar in both groups and independently associated with previous hospital attention.

Carbapenems are last-resort antibiotics used to treat multidrug-resistant bacterial infections. Resistance to carbapenems has been designated as an urgent threat and is increasing in healthcare settings. However, little is still known about the distribution and characteristics of carbapenem-resistant bacteria (CRB) outside of healthcare settings. Here, we surveyed the distribution of CRB in ten diverse freshwater and seawater environments in California, U.S., ranging from San Luis Obispo County to San Bernardino County, combining both direct isolation and enrichment approaches to increase the diversity of isolated CRB. From the locations surveyed, we selected 30 CRB for further characterization. These isolates were identified as members of the genera Aeromonas, Enterobacter, Enterococcus, Paenibacillus, Pseudomonas, Sphingobacterium, and Stenotrophomonas. These isolates were resistant to carbapenems, other β-lactams, and often to other antibiotics (tetracycline, gentamicin, or ciprofloxacin). We also found that nine isolates belonging to the genera Aeromonas, Enterobacter (blaIMI-2), and Stenotrophomonas (blaL1) produced carbapenemases. Overall, our findings indicate that sampling different types of aquatic environments and combining different isolation approaches increase the diversity of the environmental CRB obtained. Moreover, our study supports the increasingly recognized role of natural water systems as an underappreciated reservoir of bacteria resistant to carbapenems and other antibiotics, including bacteria carrying carbapenemase genes.

An increase in the carbapenem-hydrolyzing capacity of class D β-lactamase has been observed in strains of multiple species, posing a significant challenge to the control of antibiotic resistance. In this study, we aimed to investigate the genetic diversity and phylogenetic characteristics of new blaOXA-48-like variants derived from Shewanella xiamenensis. Three ertapenem-non-susceptible S. xiamenensis strains were identified, one isolated from the blood sample of an inpatient, the other two isolated from the aquatic environment. Phenotypic characterization confirmed that the strains were carbapenemase producers and exhibited antimicrobial resistance patterns to ertapenem, with some showing lower susceptibility to imipenem, chloramphenicol, ciprofloxacin, and tetracycline. No significant resistance to cephalosporins was observed. Sequence analysis revealed that one strain harbored blaOXA-181 and the other two strains harbored blaOXA-48-like genes, with open reading frame (ORF) similarities with blaOXA-48 ranging from 98.49% to 99.62%. The two novel blaOXA-48-like genes, named blaOXA-1038 and blaOXA-1039, respectively, were cloned and expressed in E. coli. The three OXA-48-like enzymes demonstrated significant hydrolysis activity against meropenem, and the classical β-lactamase inhibitor had no significant inhibitory effect. In conclusion, this study demonstrated the diversity of the blaOXA gene and highlighted the emergence of novel OXA carbapenemases in S. xiamenensis. Further attention to S. xiamenensis and OXA carbapenemases is recommended for the effective prevention and control of antibiotic-resistant bacteria. [ABSTRACT FROM AUTHOR]

The detection of KPC-type carbapenemases is necessary for guiding appropriate antibiotic therapy and the implementation of antimicrobial stewardship and infection control measures. Currently, few tests are capable of differentiating carbapenemase types, restricting the lab reports to their presence or not. The aim of this work was to raise antibodies and develop an ELISA test to detect KPC-2 and its D179 mutants. The ELISA-KPC test was designed using rabbit and mouse polyclonal antibodies. Four different protocols were tested to select the bacterial inoculum with the highest sensitivity and specificity rates. The standardisation procedure was performed using 109 previously characterised clinical isolates, showing 100% of sensitivity and 89% of specificity. The ELISA-KPC detected all isolates producing carbapenemases, including KPC variants displaying the ESBL phenotype such as KPC-33 and -66. [ABSTRACT FROM AUTHOR]

Antibiotic resistance remains one of the most pressing public health issues facing the world today. At the forefront of this battle lies the ever-increasing identification of extended-spectrum beta-lactamases and carbapenemases within human pathogens, conferring resistance towards broad-spectrum and last-resort antimicrobials. This study was prompted due to the identification of a pathogenic Aeromonas hydrophila isolate (strain MAH-4) collected from abdominal fluid, which presented a robust resistance pattern against second-, third-, and fourth-generation cephalosporins, ertapenem, ciprofloxacin, gentamicin, levofloxacin and moxifloxacin, and beta lactam/beta-lactamase inhibitor combinations. Whole genome sequencing was performed and identified a 328 kb plasmid (pMAH4) encoding 10 antibiotic resistance genes, including blaSFO-1, blaTEM-1, and blaOXA-1 of A. hydrophia MAH-4. This is the first report of beta-lactamase SFO-1 within a clinical strain of Aeromonas. Due to the remarkable sequence identity of pMAH4 to plasmids associated with Enterobacterales genera like Klebsiella and the extensive capabilities of Aeromonas for horizontal gene transfer, our identification of a clinical isolate encoding SFO-1 on a plasmid suggests antibiotic resistance gene mobility between Enterobacterales and non-Enterobacterales species.

Background: The emergence and global spread of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) are of great concern to health services worldwide. These β-lactamases hydrolyze almost all β-lactams, are plasmid-encoded, and are easily transferable among bacterial species. They are mostly of the KPC types in CR-hvKp. OXA-48-producing hvKP strains have been rarely reported in the literature. Methods: OXA-48-producing hvKP strains were collected from clinical specimens at the First Affiliated Hospital of Hainan Medical University from January 2022 to March 2023. Hypervirulent strains were tested for virulence in a mouse lethality study and underwent whole genome sequencing to identify genomic features. Results: A total of 42 unique OXA-48-bearing K. pneumoniae strains were identified, including three CR-hvKP strains (KP2683-1, NCRE61, and KP2185), which were isolated from bacteremia, pulmonary abscess, and liver abscess separately. The three CR-hvKP strains belonged to two different clones of ST11 KL64 (KP2185 and NCRE61) and ST23 K1 (KP2683-1). The KP2683-1 strain had the highest virulence. Whole genome sequencing analysis indicated that NCRE61 and KP2185 acquired IncFIB-type plasmids with a set of virulence genes (iroBCDN, iucABCD, iutA, rmpA, and rmpA2), while KP2683-1 acquired an IncL-type blaOXA-48-harboring plasmid. Consecutive cultures showed that the blaOXA-48-harboring plasmids were highly stable in the three hvKP strains and could be transmitted to Escherichia coli J53 by conjugation. The drug susceptibility testing results show that Ceftazidime/avibactam is sensitive for OXA-48-producing hvKP. Conclusions: Our study highlighted the two evolutionary pathways of OXA-48-producing hvKP strains and confirmed their virulence through in vivo testing. Ceftazidime/avibactam may be a viable option for treating OXA-48-producing hvKP strains.

Multidrug-resistant (MDR) and extended spectrum β-lactamase (ESBL)-producing extra-intestinal K. pneumoniae are associated with increased morbidity and mortality. This study aimed to characterize the resistance and virulence profiles of extra-intestinal MDR ESBL-producing K. pneumoniae associated with infections at a tertiary hospital in South-Kivu province, DRC. Whole-genome sequencing (WGS) was carried out on 37 K. pneumoniae isolates displaying MDR and ESBL-producing phenotype. The assembled genomes were analysed for phylogeny, virulence factors and antimicrobial resistance genes (ARG) determinants. These isolates were compared to sub-Saharan counterparts. K. pneumoniae isolates displayed a high genetic variability with up to 16 sequence types (ST). AMR was widespread against β-lactamases (including third and fourth-generation cephalosporins, but not carbapenems), aminoglycosides, ciprofloxacin, tetracycline, erythromycin, nitrofurantoin, and cotrimoxazole. The blaCTX-M-15 gene was the most common β-lactamase gene among K. pneumoniae isolates. No carbapenemase gene was found. ARG for aminoglycosides, quinolones, phenicols, tetracyclines, sulfonamides, nitrofurantoin were widely distributed among the isolates. Nine isolates had the colistin-resistant R256G substitution in the pmrB efflux pump gene without displaying reduced susceptibility to colistin. Despite carrying virulence genes, none had hypervirulence genes. Our results highlight the genetic diversity of MDR ESBL-producing K. pneumoniae isolates and underscore the importance of monitoring simultaneously the evolution of phenotypic and genotypic AMR in Bukavu and DRC, while calling for caution in administering colistin and carbapenem to patients. [ABSTRACT FROM AUTHOR]

Background: Klebsiella pneumoniae, a member of the ESKAPE group of bacterial pathogens, has developed multi-antimicrobial resistance (AMR), including resistance to carbapenems, which has increased alarmingly due to the acquisition of carbapenemase genes located on specific plasmids. Methods: Four clinical K. pneumoniae isolates were collected from four patients of a neuro-intensive care unit in Moscow, Russia, during the point prevalence survey. The AMR phenotype was estimated using the Vitec-2 instrument, and whole genome sequencing (WGS) was done using Illumina and Nanopore technologies. Results: All strains were resistant to beta-lactams, nitrofurans, fluoroquinolones, sulfonamides, aminoglycosides, and tetracyclines. WGS analysis revealed that all strains were closely related to K. pneumoniae ST39, capsular type K-23, with 99.99% chromosome identity. The novelty of the study is the description of the strains carrying simultaneously three large plasmids of the IncHI1B, IncC, and IncFIB groups carrying the carbapenemase genes of three types, blaOXA-48, blaNDM-1, and blaKPC-2, respectively. The first of them, highly identical in all strains, was a hybrid plasmid that combined two regions of the resistance genes (blaOXA-48 and blaTEM-1 + blaCTX-M-15 + blaOXA-1 + catB + qnrS1 + int1) and a region of the virulence genes (iucABCD, iutA, terC, and rmpA2::IS110). Conclusion: The spread of K. pneumoniae strains carrying multiple plasmids conferring resistance even to last-resort antibiotics is of great clinical concern. [ABSTRACT FROM AUTHOR]

Bivalves are filter-feeding organisms and biomarkers of bacterial pollution. Our study aimed to analyze the occurrence and characteristics of extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing Escherichia coli among bivalves. A total of 522 bivalve samples were collected along Portuguese shellfish production areas. Homogenized samples were screened for E. coli contamination on corresponding selective plates, allowing for concomitant growth of Klebsiella pneumoniae. E. coli growth was observed in 39% of the samples. Subsequent selective screening identified nine samples (4.4%) contaminated with ESBL producers, corresponding to E. coli (n = 7) and K. pneumoniae (n = 2), while a single carbapenemase-producing K. pneumoniae (0.5%) was identified. ESBLs were all CTX-M-types commonly identified in human isolates, i.e., CTX-M-32 (n = 4), CTX-M-15 (n = 4), and CTX-M-14 (n = 1). The carbapenemase producer harbored the blaGES-5 gene located on a ColE plasmid. Clonality was evaluated by multilocus sequence typing, identifying E. coli backgrounds as ST10, ST23, ST540, ST617, ST746, SLV206, and SLV2325, commonly identified among environmental and human strains. The K. pneumoniae isolates belonged to ST834, ST15, and DLV644. The occurrence of ESBL- and carbapenemase-producing Enterobacteriaceae in bivalves reveals how the marine environment constitutes a reservoir of critical bacterial pathogens, thus potentially representing a risk to human health. [ABSTRACT FROM AUTHOR]

Patients diagnosed with acute leukemia (AL) have a weakened immune system. Infections acquired by these patients are cause for concern and especially worrisome when Gram-negative multidrug-resistant (MDR) bacteria are involved, as they are difficult to treat, especially in the case of ESBL- and/or carbapenemase-producing Enterobacterales. Culture-based approaches have been relied on over the past decades as the method of choice for the early detection of gut colonization by MDR Gram-negative bacteria. However, various studies have indicated its limited sensitivity, underlining the need for new screening procedures in onco-hematological patients. Here, we evaluated a shotgun metagenomics approach to detect ESBL- and/or carbapenemase-producing Enterobacterales in the gut of 28 patients who had recovered from AL, which were previously colonized by these bacteria but cured at the time of sampling, as judged by culture-based methods. No ESBL or carbapenemase determinants were detected among the many resistance genes found by the metagenomics approach, supporting that patients were truly decolonized, with considerable consequences for their future clinical management. Due to the relatively low number of patients available for the present investigation, further studies should be conducted to support the utility and applicability of metagenomics for the routine screening of MDR bacteria in onco-hematological patients. [ABSTRACT FROM AUTHOR]

Carbapenems are atypical β-lactam antibiotics with a broade antibacterial spectrum and strong antibacterial activity; however, the emergence and spread of carbapenemases have led to a decline in their effectiveness. New Delhi metallo-β-lactamase (NDM) is an important carbapenemase that has attracted widespread attention and poses a major threat to public health. To investigate the epidemiological characteristics of blaNDM in swine and chicken farms in southwestern China, we isolated 102 blaNDM-positive Enterobacterales strains from 18 farms in Sichuan and Yunnan provinces in 2021, with Escherichia coli and Klebsiella spp. being the main reservoirs of blaNDM, variant blaNDM-5 being the most prevalent, and all strains being multi-drug resistant. Whole-genome sequencing analysis of 102 blaNDM-positive Enterobacterales strains revealed that blaNDM had spread primarily through its carriers on the same farm and among the 18 farms in this study. A high degree of genetic similarity between animal-derived blaNDM-positive Escherichia coli strains and human-derived strains was also identified, suggesting a potential mutual transmission between them. Nanopore sequencing results indicated that blaNDM is predominantly present on the IncX3 plasmid, that an insertion sequence might be important for recombination in the blaNDM genetic environment, and that most of the plasmids carrying blaNDM are transferable. Collectively, our results enrich the current epidemiological information regarding blaNDM in pig and chicken farms in Southwest China, revealing its transmission pattern, as well as the potential risk of transmission to humans, which could help to better understand and control the spread of blaNDM.

The gastrointestinal tract is an important reservoir of high-risk Enterobacteria clones and a driver of antimicrobial resistance in hospitals. In this study, patients from six hospitals in four major Bulgarian towns were included in this study. Overall, 205 cefotaxime-resistant isolates (35.3%) of Enterobacterales order were detected in fecal samples among 580 patients during the period of 2017–2019. ESBL/carbapenemase/plasmidic AmpC producer rates were 28.8%, 2.4%, and 1.2%, respectively. A wide variety of ESBLs: CTX-M-15 (41%), CTX-M-3 (24%), CTX-M-27 (11%), and CTX-M-14 (4%) was found. The carbapenemases identified in this study were New Delhi metalo-β-lactamase (NDM)-1 (5.4%) and Klebsiella carbapenemase (KPC)-2 (1.5%). Most NDM-1 isolates also produced CTX-M-15/-3 and CMY-4 β-lactamases. They belonged to ST11 Klebsiella pneumoniae clone. The epidemiology typing revealed three main high-risk K. pneumoniae clones (26%)—ST11, ST258, and ST15 and five main Escherichia coli clones—ST131 (41.7%), ST38, ST95, ST405, and ST69. Sixty-one percent of ST131 isolates were from the highly virulent epidemic clone O25b:H4-ST131. Phylotyping revealed that 69% of E. coli isolates belonged to the virulent B2 and D groups. Almost all (15/16) Enterobacter isolates were identified as E. hormaechei and the most common ST type was ST90. Among all of the isolates, a high ESBL/carbapenemases/plasmid AmpC (32.4%) prevalence was observed. A significant proportion of the isolates (37%) were members of high-risk clones including two pan-drug-resistant K. pneumoniae ST11 NDM-1 producing isolates. Due to extensive antibiotic usage during COVID-19, the situation may worsen, so routine screenings and strict infection control measures should be widely implemented. [ABSTRACT FROM AUTHOR]

The emergence of carbapenem-resistant Enterobacterales (CRE) has been recognized as a significant concern globally. Ceftazidime/avibactam (CZA) is a novel β-lactam/β-lactamase inhibitor that has demonstrated activity against isolates producing class A, C, and D β-lactamases. Here-in, we evaluated the in vitro activity of CZA and comparator antimicrobial agents against 858 CRE isolates, arising from the Southeast Asian region, collected from a large tertiary hospital in Singapore. These CRE isolates mainly comprised Klebsiella pneumoniae (50.5%), Escherichia coli (29.4%), and Enterobacter cloacae complex (17.1%). Susceptibility rates to levofloxacin, imipenem, meropenem, doripenem, aztreonam, piperacillin/tazobactam, cefepime, tigecycline, and polymyxin B were low. CZA was the most active β-lactam agent against 68.9% of the studied isolates, while amikacin was the most active agent among all comparator antibiotics (80% susceptibility). More than half of the studied isolates (51.4%) identified were Klebsiella pneumoniae carbapenemase (KPC)-2 producers, 25.9% were New Delhi metallo-β-lactamase (NDM) producers, and Oxacillinase (OXA)-48-like producers made up 10.7%. CZA was the most active β-lactam agent against KPC-2, OXA-48-like, and Imipenemase (IMI) producers (99.3% susceptible; MIC50/90: ≤1/2 mg/L). CZA had excellent activity against the non-carbapenemase-producing CRE (91.4% susceptible; MIC50/90: ≤1/8 mg/L). Expectedly, CZA had no activity against the metallo-β-lactamases (MBL)-producing CRE (NDM- and Imipenemase MBL (IMP) producers; 27.2% isolates), and the carbapenemase co-producing CRE (NDM + KPC, NDM + OXA-48-like, NDM + IMP; 3.0% isolates). CZA is a promising addition to our limited armamentarium against CRE infections, given the reasonably high susceptibility rates against these CRE isolates. Careful stewardship and rational dosing regimens are required to preserve CZA’s utility against CRE infections.

The spread of carbapenemase-producing (CP) Enterobacterales is currently a worldwide concern, especially in the elderly. Twelve CP-E. coli isolated from rectal swabs of colonized inpatients aged ≥65 years from four hospitals in two Italian cities (Milan and Rome) were analyzed by whole genome sequencing (WGS) to obtain multi-locus sequence typing (MLST), identification of carbapenemase-encoding genes, resistome, plasmid content, and virulence genes. MLST analysis showed the presence of 10 unrelated lineages: ST410 (three isolates from three different hospitals in two cities) and ST12, ST38, ST69, ST95, ST131, ST189, ST648, ST1288, and ST1598 (one isolate each). Most isolates (9/12, 75%) contained a serine-β-lactamase gene (5 blaKPC-3, 2 blaKPC-2, and 2 blaOXA-181), while three isolates harbored a metallo-β-lactamase gene (two blaNDM-5 and one blaVIM-1). In most CP-E. coli, the presence of more than one plasmid was observed, with the predominance of IncF. Several virulence genes were detected. All isolates contained genes enhancing the bacterial fitness, such as gad and terC, and all isolates but one, fimH, encoding type 1 fimbriae. In conclusion, CP-E. coli clones colonizing elderly patients showed heterogeneous genetic backgrounds. We recommend strict surveillance to monitor and prevent the spread of successful, high-risk clones in healthcare settings. [ABSTRACT FROM AUTHOR]

This study aimed to investigate, for the first time, the occurrence and characteristics of extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing Enterobacterales in Cape Verde. A total of 98 inpatients hospitalized at Hospital Universitário Agostinho Neto were screened for rectal colonization. All ESBL- and carbapenemase-producing isolates were tested for antimicrobial susceptibility and characterized by multilocus sequence typing. Mating-out assay followed by PCR-based replicon typing were performed to characterize the plasmids harboring carbapenemase encoding genes. A large proportion of patients carried ESBL- or carbapenemase-producing Enterobacterales (56% and 6%, respectively). Among 93 ESBL-producing isolates, there were mainly Klebsiella pneumoniae (58%) and Escherichia coli (37%). Five different ESBLs were detected, with CTX-M-15 being highly predominant (92%). Six carbapenemase-producing isolates (five E. coli and one K. pneumoniae) were recovered, and all of the OXA-48-like type (four OXA-181, one OXA-48, and one OXA-244). The blaOXA-48 gene was located on an IncFI-type plasmid, the blaOXA-181 gene on IncFI or IncX3 plasmids, and the blaOXA-244 gene was found to be chromosomally located. The five carbapenemase-producing E. coli isolates belonged to five distinct sequence types. This study overall showed a very high prevalence of ESBL-producing Enterobacterales, as well as the emergence of carbapenemase producers in this hospital. [ABSTRACT FROM AUTHOR]

Antibiotic-resistant bacteria, and more specifically, carbapenem-producing Enterobacterales (CPE) strains, are increasing worldwide. Despite their growing prevalence, in most high-income countries, the detection of CPE is still considered a low-frequency event. Sporadically, patients co-colonized with distinct CPE strains and/or different carbapenemase enzymes are detected. In this paper, we present three cases that illustrate the underlying mechanisms of co-colonization, focusing on horizontal gene transfer (HGT) and patient-to-patient transmission. We also demonstrate the diversity of CPE species and discuss the potential consequences of co-colonization. [ABSTRACT FROM AUTHOR]

Background: Acinetobacter baumannii is a common cause of multi-drug (MDR)-resistant infections worldwide. The epidemiological and molecular characteristics of MDR-A. baumannii in Jordan is not known. Methods: A. baumannii isolates were collected from 2010 to 2020 from three tertiary hospitals in Jordan. Demographic and clinical data, isolates information, antibiotic susceptibility patterns, phenotypic, and molecular characterization of carbapenem resistance genes were performed. Results: A total of 622 A. baumannii isolates were collected during the study period. Most isolates were from males, aged 18–60 years, Jordanian, from infected wounds, and were patients in surgery or critical care units. Among patients from whom A. baumannii was isolated, associated risk factors for MDR were adults over 60, males, critically ill patients and infected wounds (OR 4.14, 2.45, 10, 7, respectively, p < 0.0001). Incidence rates from 2010 to 2015 showed a slight increase in MDR (3.75/1000 to 4.46/1000). Resistance patterns indicated high resistance for most cephalosporins, carbapenems, and fluoroquinolones, moderate resistance for trimethoprim/sulfamethoxazole and ampicillin/sulbactam, low resistance for aminoglycosides and tetracyclines, while colistin and tigecycline, have the lowest resistance rates. 76.8% of A. baumannii isolates were MDR and 99.2% were carbapenem-resistant. All isolates were positive for the OXA-51 gene (100%), 98.5% were positive for the OXA-23 gene, 26.6% for the VIM gene, while KPC and IMP genes were almost not detected (0% and 0.8% respectively). Conclusions: This is the first large, multicentric, prolonged study that provides insights into A. baumannii infections in Jordan. Attention to patients at higher risk is important for early identification. Colistin and tigecycline were the most effective antimicrobials. [ABSTRACT FROM AUTHOR]

Aminoglycosides (AGs) in combination with β-lactams play an important role in antimicrobial therapy in severe infections. Pan-resistance to clinically relevant AGs increasingly arises from the production of 16S rRNA methylases (RMTases) that are mostly encoded by plasmids in Gram-negative bacteria. The recent emergence and spread of isolates encoding RMTases is worrisome, considering that they often co-produce extended-spectrum β-lactamases (ESBLs) or carbapenemases. Our study aimed to retrospectively analyze and characterize the association of carbapenem- and aminoglycoside-resistant clinical isolates in Switzerland during a 3.5-year period between January 2017 and June 2020. A total of 103 pan-aminoglycoside- and carbapenem-resistant clinical isolates were recovered at the NARA (Swiss National Reference Center for Emerging Antibiotic Resistance) during the 2017–2020 period. Carbapenemase and RMTase determinants were identified by PCR and sequencing. The characterization of plasmids bearing resistance determinants was performed by a mating-out assay followed by PCR-based replicon typing (PBRT). Clonality of the isolates was investigated by multilocus sequence typing (MLST). Over the 991 Enterobacterales collected at the NARA during this period, 103 (10.4%) of them were resistant to both carbapenems and all aminoglycosides. Among these 103 isolates, 35 isolates produced NDM-like carbapenemases, followed by OXA-48-like (n = 23), KPC-like (n = 21), or no carbapenemase (n = 13), OXA-48-like and NDM-like co-production (n = 7), and VIM-like enzymes (n = 4). The RMTases ArmA, RmtB, RmtC, RmtF, RmtG, and RmtB + RmtF were identified among 51.4%, 13.6%, 4.9%, 24.3%, 1%, and 1%, respectively. Plasmid co-localization of the carbapenemase and the RMTase encoding genes was found among ca. 20% of the isolates. A high diversity was identified in terms of the nature of associations between RMTase and carbapenemase-encoding genes, of incompatibility groups of the corresponding plasmids, and of strain genetic backgrounds, highlighting heterogeneous importations rather than clonal dissemination. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant pathogens mediated by metallo-beta-lactamases (MBLs) have spread worldwide, where NDM-1 is a typical and key MBL. Here, we firstly discussed the distribution characterization of NDM-1, which produces multidrug-resistant Proteus mirabilis among broilers in China. From January to April 2019, 40 (18.1%, 40/221) blaNDM-1-carrying P. mirabilis strains were recovered from commercial broilers in slaughterhouse B in China. All the isolates were resistant to imipenem, meropenem and other β-lactams. These isolates belong to five clusters identified via pulsed field gel electrophoresis (PFGE). Further studies on twenty representative strains revealed that seven blaNDM-1 genes were located on plasmids with sizes of 104.5–138.9 kb. Notably, only three strains (PB72, PB96 and PB109) were successfully transferred to Escherichia coli J53, while the other four isolates were located in nontransferable plasmids. The rest were harbored in chromosomes. Ulteriorly, based on whole genome sequencing (WGS), these twenty isolates showed four typical phylogenetic clades according to single nucleotide polymorphisms (SNPs) of a core genome and presented four main genomic backbone profiles, in which type II/III strains shared a similar genetic context. All of the above is evidence of blaNDM-1 transmission and evolution in P. mirabilis, suggesting that the prevalence may be more diverse in broiler farms. Accordingly, as intestinal and environmental symbiotic pathogens, blaNDM-1-positive P. mirabilis will pose greater threats to the environment and public health. [ABSTRACT FROM AUTHOR]

Patients diagnosed with acute leukemia (AL) have a weakened immune system. Infections acquired by these patients are cause for concern and especially worrisome when Gram-negative multidrug-resistant (MDR) bacteria are involved, as they are difficult to treat, especially in the case of ESBL- and/or carbapenemase-producing Enterobacterales. Culture-based approaches have been relied on over the past decades as the method of choice for the early detection of gut colonization by MDR Gram-negative bacteria. However, various studies have indicated its limited sensitivity, underlining the need for new screening procedures in onco-hematological patients. Here, we evaluated a shotgun metagenomics approach to detect ESBL- and/or carbapenemase-producing Enterobacterales in the gut of 28 patients who had recovered from AL, which were previously colonized by these bacteria but cured at the time of sampling, as judged by culture-based methods. No ESBL or carbapenemase determinants were detected among the many resistance genes found by the metagenomics approach, supporting that patients were truly decolonized, with considerable consequences for their future clinical management. Due to the relatively low number of patients available for the present investigation, further studies should be conducted to support the utility and applicability of metagenomics for the routine screening of MDR bacteria in onco-hematological patients.

Background: Klebsiella pneumoniae, a member of the ESKAPE group of bacterial pathogens, has developed multi-antimicrobial resistance (AMR), including resistance to carbapenems, which has increased alarmingly due to the acquisition of carbapenemase genes located on specific plasmids. Methods: Four clinical K. pneumoniae isolates were collected from four patients of a neuro-intensive care unit in Moscow, Russia, during the point prevalence survey. The AMR phenotype was estimated using the Vitec-2 instrument, and whole genome sequencing (WGS) was done using Illumina and Nanopore technologies. Results: All strains were resistant to beta-lactams, nitrofurans, fluoroquinolones, sulfonamides, aminoglycosides, and tetracyclines. WGS analysis revealed that all strains were closely related to K. pneumoniae ST39, capsular type K-23, with 99.99% chromosome identity. The novelty of the study is the description of the strains carrying simultaneously three large plasmids of the IncHI1B, IncC, and IncFIB groups carrying the carbapenemase genes of three types, blaOXA-48, blaNDM-1, and blaKPC-2, respectively. The first of them, highly identical in all strains, was a hybrid plasmid that combined two regions of the resistance genes (blaOXA-48 and blaTEM-1 + blaCTX-M-15 + blaOXA-1 + catB + qnrS1 + int1) and a region of the virulence genes (iucABCD, iutA, terC, and rmpA2::IS110). Conclusion: The spread of K. pneumoniae strains carrying multiple plasmids conferring resistance even to last-resort antibiotics is of great clinical concern.

The spread of carbapenemase-producing (CP) Enterobacterales is currently a worldwide concern, especially in the elderly. Twelve CP-E. coli isolated from rectal swabs of colonized inpatients aged ≥65 years from four hospitals in two Italian cities (Milan and Rome) were analyzed by whole genome sequencing (WGS) to obtain multi-locus sequence typing (MLST), identification of carbapenemase-encoding genes, resistome, plasmid content, and virulence genes. MLST analysis showed the presence of 10 unrelated lineages: ST410 (three isolates from three different hospitals in two cities) and ST12, ST38, ST69, ST95, ST131, ST189, ST648, ST1288, and ST1598 (one isolate each). Most isolates (9/12, 75%) contained a serine-β-lactamase gene (5 blaKPC-3, 2 blaKPC-2, and 2 blaOXA-181), while three isolates harbored a metallo-β-lactamase gene (two blaNDM-5 and one blaVIM-1). In most CP-E. coli, the presence of more than one plasmid was observed, with the predominance of IncF. Several virulence genes were detected. All isolates contained genes enhancing the bacterial fitness, such as gad and terC, and all isolates but one, fimH, encoding type 1 fimbriae. In conclusion, CP-E. coli clones colonizing elderly patients showed heterogeneous genetic backgrounds. We recommend strict surveillance to monitor and prevent the spread of successful, high-risk clones in healthcare settings.

Background: Acinetobacter baumannii is a common cause of multi-drug (MDR)-resistant infections worldwide. The epidemiological and molecular characteristics of MDR-A. baumannii in Jordan is not known. Methods: A. baumannii isolates were collected from 2010 to 2020 from three tertiary hospitals in Jordan. Demographic and clinical data, isolates information, antibiotic susceptibility patterns, phenotypic, and molecular characterization of carbapenem resistance genes were performed. Results: A total of 622 A. baumannii isolates were collected during the study period. Most isolates were from males, aged 18–60 years, Jordanian, from infected wounds, and were patients in surgery or critical care units. Among patients from whom A. baumannii was isolated, associated risk factors for MDR were adults over 60, males, critically ill patients and infected wounds (OR 4.14, 2.45, 10, 7, respectively, p < 0.0001). Incidence rates from 2010 to 2015 showed a slight increase in MDR (3.75/1000 to 4.46/1000). Resistance patterns indicated high resistance for most cephalosporins, carbapenems, and fluoroquinolones, moderate resistance for trimethoprim/sulfamethoxazole and ampicillin/sulbactam, low resistance for aminoglycosides and tetracyclines, while colistin and tigecycline, have the lowest resistance rates. 76.8% of A. baumannii isolates were MDR and 99.2% were carbapenem-resistant. All isolates were positive for the OXA-51 gene (100%), 98.5% were positive for the OXA-23 gene, 26.6% for the VIM gene, while KPC and IMP genes were almost not detected (0% and 0.8% respectively). Conclusions: This is the first large, multicentric, prolonged study that provides insights into A. baumannii infections in Jordan. Attention to patients at higher risk is important for early identification. Colistin and tigecycline were the most effective antimicrobials.

Carbapenemase-KPC producing Klebsiella pneumoniae (CP-Kp) infection represents a serious threat to solid organ transplant (SOT). All patients admitted between 1 May 2011 and 31 August 2014 undergoing SOT were included in the retrospective study. The primary outcomes included a description of the association of enteric colonization and invasive infections by CP-Kp with one-year mortality. Secondary outcomes were the study of risk factors for colonization and invasive infections by CP-Kp. Results: A total of 5.4% (45/828) of SOT recipients had at least one positive rectal swab for CP-Kp, with most (88.9%) occurring after transplantation. 4.5% (35/828) of patients developed a CP-Kp-related invasive infection, with 68.6% (24/35) being previously colonized. The 1-year mortality was 31.1% in patients with enteric colonization with CP-Kp and, it was 51.4% among patients with CP-Kp-related invasive infections. At univariate analysis, colonization, invasive infections, sepsis, severe sepsis, and septic shock were significantly associated with 1-year mortality. At multivariate analysis, only invasive infections and the combination of sepsis, severe sepsis, or septic shock were significantly associated with 1-year mortality, whereas gastrointestinal colonization was significantly associated with survival. In this population, the 1-year mortality was significantly associated with invasive infections; otherwise, gastrointestinal colonization was not associated with increased 1-year mortality.

Using whole-genome sequencing and cloning of the target gene, we identified blaOXA-900 carbapenemase, a novel blaOXA belonging to a distant and distinct sub-family of blaOXA-48-like. The plasmid-mediated gene was identified in a C. freundii isolate with elevated carbapenem MICs that evaded detection by commercial DNA-based methods. The novel gene, an OXA-48 family carbapenem-hydrolyzing class D β-lactamase, OXA-900, likely originates from marine environmental Shewanella. Since this plasmid-mediated gene has entered a member of the Enterobacterales and evades detection by commonly used tests, it may gain wide dissemination among Enterobacterales.

Background: The emergence and global spread of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) are of great concern to health services worldwide. These β-lactamases hydrolyze almost all β-lactams, are plasmid-encoded, and are easily transferable among bacterial species. They are mostly of the KPC types in CR-hvKp. OXA-48-producing hvKP strains have been rarely reported in the literature., Methods: OXA-48-producing hvKP strains were collected from clinical specimens at the First Affiliated Hospital of Hainan Medical University from January 2022 to March 2023. Hypervirulent strains were tested for virulence in a mouse lethality study and underwent whole genome sequencing to identify genomic features., Results: A total of 42 unique OXA-48-bearing K. pneumoniae strains were identified, including three CR-hvKP strains (KP2683-1, NCRE61, and KP2185), which were isolated from bacteremia, pulmonary abscess, and liver abscess separately. The three CR-hvKP strains belonged to two different clones of ST11 KL64 (KP2185 and NCRE61) and ST23 K1 (KP2683-1). The KP2683-1 strain had the highest virulence. Whole genome sequencing analysis indicated that NCRE61 and KP2185 acquired IncFIB-type plasmids with a set of virulence genes ( iroBCDN , iucABCD , iutA , rmpA , and rmpA2 ), while KP2683-1 acquired an IncL-type bla OXA-48 -harboring plasmid. Consecutive cultures showed that the bla OXA-48 -harboring plasmids were highly stable in the three hvKP strains and could be transmitted to Escherichia coli J53 by conjugation. The drug susceptibility testing results show that Ceftazidime/avibactam is sensitive for OXA-48-producing hvKP., Conclusions: Our study highlighted the two evolutionary pathways of OXA-48-producing hvKP strains and confirmed their virulence through in vivo testing. Ceftazidime/avibactam may be a viable option for treating OXA-48-producing hvKP strains.

Due to the increasing reports of carbapenemase-producing Enterobacteriaceae (CPE) from livestock in recent years, the European Reference Laboratory for Antimicrobial Resistances (EURL-AR) provided a protocol for their recovery from caecum and meat samples. This procedure exhibited limitations for the detection of CPE with low carbapenem MIC values. Therefore, it was modified by a second, selective enrichment in lysogeny broth with cefotaxime (CTX 1 mg/L) and with meropenem (MEM 0.125 mg/L) at 37 °C under microaerophilic conditions. By Real-time PCR, these enrichments are pre-screened for the most common carbapenemase genes. Another adaptation was the use of in-house prepared MacConkey agar with MEM and MEM+CTX instead of commercial selective agar. According to the EURL-method, we achieved 100% sensitivity and specificity using the in-house media instead of commercial agar, which decreased the sensitivity to ~75%. Comparing the method with and without the second enrichment, no substantial influence on sensitivity and specificity was detected. Nevertheless, this enrichment has simplified the CPE-isolation regarding the accompanying microbiota and the separation of putative colonies. In conclusion, the sensitivity of the method can be increased with slight modifications.

Urinary tract infections (UTIs) are common in clinics and hospitals and are associated with a high economic burden. Enterobacterium Klebsiella pneumoniae is a prevalent agent causing UTIs. A high prevalence of carbapenem-resistant K. pneumoniae (CRKP) has emerged recently and is continuing to increase. Seventeen urinary CRKP isolates collected at a teaching hospital in Taiwan from December 2016 to September 2017 were analyzed to elucidate their drug resistance mechanisms. Two-thirds of the isolates were obtained from outpatients. Antimicrobial susceptibility tests demonstrated multidrug resistance in all the isolates. Multilocus sequence typing analysis showed high diversity among the isolates. PCR analysis demonstrated the presence of carbapenemases in three isolates. All isolates carried at least one other extended-spectrum β-lactamase, including TEM, DHA, and CTX-M. Fifteen isolates contained mutations in one of the outer membrane porins that were assessed. The expression levels of the acrB and/or oqxB efflux pump genes, as determined by qRT-PCR, were upregulated in 11 isolates. Six isolates might have utilized other efflux pumps or antimicrobial resistance mechanisms. These analyses demonstrated a highly diverse population and the presence of complex resistance mechanisms in urinary isolates of K. pneumoniae.

Background: Carbapenemase-producing Enterobacteriaceae (CPE) are an emerging threat in healthcare settings worldwide. Objectives: We evaluated the presence of carbapenemase genes in CPE in a tertiary care university hospital in Tokyo, Japan. Methods: Carbapenem-resistant clinical isolates were collected in 2018 at Teikyo University Hospital (Tokyo, Japan). Bacterial species were identified using MALDI-TOF MS. Carbapenemase production was evaluated using a carbapenemase inactivation method. The presence of carbapenemase genes was confirmed by multiplex PCR and DNA sequencing. Results: Four CPE isolates were identified: two Enterobacter cloacae complex strains and Klebsiella oxytoca and Klebsiella pneumoniae strains. Three of the isolates (E. cloacae complex and K. oxytoca) were IMP-1-type producers, including IMP-10 in their produced metallo-β-lactamase, and are epidemic in East Japan. The IMP-10-producing E. cloacae complex strain also produced CTX-M ESBL. The other CPE isolate (K. pneumoniae) is a VIM-1 producer. VIM-1-producing K. pneumoniae is epidemic in Europe, especially in Greece. Accordingly, the VIM-1 producer was isolated from a patient with a medical history in Greece. Conclusions: This study revealed the emergence of E. cloacae complex co-producing IMP-1-type carbapenemase and CTX-M ESBL, and K. pneumoniae producing VIM-1 carbapenemase in clinical isolates in Japan. Metallo-β-lactamase was the most prevalent type of carbapenemase at Teikyo University Hospital, especially IMP-1-type carbapenemase. The detection of VIM-1-producing K. pneumoniae suggests that epidemic CPE from overseas can spread to countries with low CPE prevalence, such as Japan, highlighting the need for active surveillance.

Resistance to carbapenems due to carbapenemase-producing Enterobacteriaceae (CPE) is an increasing threat to human health worldwide. In recent years, CPE could be found only sporadically from livestock, but concern rose that livestock might become a reservoir for CPE. In 2019, the first GES carbapenemase-producing Escherichia coli from livestock was detected within the German national monitoring on antimicrobial resistance. The isolate was obtained from pig feces and was phenotypically resistant to meropenem and ertapenem. The isolate harbored three successive blaGES genes encoding for GES-1, GES-5 and GES-5B in an incomplete class-I integron on a 12 kb plasmid (pEC19-AB02908; Acc. No. MT955355). The strain further encoded for virulence-associated genes typical for uropathogenic E. coli, which might hint at an increased pathogenic potential. The isolate produced the third carbapenemase detected from German livestock. The finding underlines the importance CPE monitoring and detailed characterization of new isolates.

In order to evaluate whether seagulls living on the Lisbon coastline, Portugal, might be colonized and consequently represent potential spreaders of multidrug-resistant bacteria, a total of 88 gull fecal samples were screened for detection of extended-spectrum β-lactamase (ESBL)- or carbapenemase-producing Enterobacteriaceae for methicillin-resistant Staphylococcus aureus (MRSA) and for vancomycin-resistant Enterococci (VRE). A large proportion of samples yielded carbapenemase- or ESBL-producing Enterobacteriaceae (16% and 55%, respectively), while only two MRSA and two VRE were detected. Mating-out assays followed by PCR and whole-plasmid sequencing allowed to identify carbapenemase and ESBL encoding genes. Among 24 carbapenemase-producing isolates, there were mainly Klebsiella pneumoniae (50%) and Escherichia coli (33%). OXA-181 was the most common carbapenemase identified (54%), followed by OXA-48 (25%) and KPC-2 (17%). Ten different ESBLs were found among 62 ESBL-producing isolates, mainly being CTX-M-type enzymes (87%). Co-occurrence in single samples of multiple ESBL- and carbapenemase producers belonging to different bacterial species was observed in some cases. Seagulls constitute an important source for spreading multidrug-resistant bacteria in the environment and their gut microbiota a formidable microenvironment for transfer of resistance genes within bacterial species.

Resistance to carbapenems is a severe threat to human health. These last resort antimicrobials are indispensable for the treatment of severe human infections with multidrug-resistant Gram-negative bacteria. In accordance with their increasing medical impact, carbapenemase-producing Enterobacteriaceae (CPE) might be disseminated from colonized humans to non-human reservoirs (i.e., environment, animals, food). In Germany, the occurrence of CPE in livestock and food has been systematically monitored since 2016. In the 2019 monitoring, an OXA-48-producing E. coli (19-AB01443) was recovered from a fecal sample of a fattening pig. Phenotypic resistance was confirmed by broth microdilution and further characterized by PFGE, conjugation, and combined short-/long-read whole genome sequencing. This is the first detection of this resistance determinant in samples from German meat production. Molecular characterization and whole-genome sequencing revealed that the blaOXA-48 gene was located on a common pOXA-48 plasmid-prototype. This plasmid-type seems to be globally distributed among various bacterial species, but it was frequently associated with clinical Klebsiella spp. isolates. Currently, the route of introduction of this plasmid/isolate combination into the German pig production is unknown. We speculate that due to its strong correlation with human isolates a transmission from humans to livestock has occurred.

Whole-genome sequence (WGS) analyses were employed to investigate the genomic epidemiology of extensively drug-resistant Klebsiella pneumoniae strains, focusing on the carbapenem resistance-encoding determinants, mobile genetic support, clonal and epidemiological relationships. A total of ten isolates were obtained from patients admitted to the intensive care unit (ICU) in a public hospital in South Africa. Five isolates were from rectal swabs of colonized patients and five from blood cultures of patients with invasive carbapenem-resistant infections. Following microbial identification and antibiotic susceptibility tests, the isolates were subjected to WGS on the Illumina MiSeq platform. All the isolates showed genotypic resistance to tested β-lactams (NDM-1, OXA-1, CTX-M-15, TEM-1B, SHV-1) and other antibiotics. All but one isolate belonged to the ST152 with a novel sequence type, ST3136, differing by a single-locus variant. The isolates had the same plasmid multilocus sequence type (IncF[K12:A-:B36]) and capsular serotype (KL149), supporting the epidemiological linkage between the clones. Resistance to carbapenems in the 10 isolates was conferred by the blaNDM-1 mediated by the acquisition of multi-replicon [ColRNAI, IncFIB(pB171), Col440I, IncFII, IncFIB(K) and IncFII(Yp)] p18-43_01 plasmid. These findings suggest that the acquisition of blaNDM-1-bearing plasmid structure (p18-43_01), horizontal transfer and clonal dissemination facilitate the spread of carbapenemases in South Africa. This emphasizes the importance of targeted infection control measures to prevent dissemination.

Preventing the spread of multidrug-resistant Gram-negative bacteria (MDRGNB) is a public health priority. However, the definition of MDRGNB applied for planning infection prevention measures such as barrier precautions differs depending on national guidelines. This is particularly relevant in the Dutch-German border region, where patients are transferred between healthcare facilities located in the two different countries, because clinicians and infection control personnel must understand antibiograms indicating MDRGNB from both sides of the border and using both national guidelines. This retrospective study aimed to compare antibiograms of Gram-negative bacteria and classify them using the Dutch and German national standards for MDRGNB definition. A total of 31,787 antibiograms from six Dutch and four German hospitals were classified. Overall, 73.7% were no MDRGNB according to both guidelines. According to the Dutch and German guideline, 7772/31,787 (24.5%) and 4586/31,787 (12.9%) were MDRGNB, respectively (p < 0.0001). Major divergent classifications were observed for extended-spectrum β-lactamase (ESBL) -producing Enterobacteriaceae, non-carbapenemase-producing carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Stenotrophomonas maltophilia. The observed differences show that medical staff must carefully check previous diagnostic findings when patients are transferred across the Dutch-German border, as it cannot be assumed that MDRGNB requiring special hygiene precautions are marked in the transferred antibiograms in accordance with both national guidelines. [ABSTRACT FROM AUTHOR]

Virulence plasmids are associated with hypervirulent types of Klebsiella pneumoniae, which generally do not carry antibiotic resistance genes. In contrast, nosocomial isolates are often associated with resistance, but rarely with virulence plasmids. Here, we describe virulence plasmids in nosocomial isolates of “high-risk” clones of sequence types (STs) 15, 48, 101, 147 and 383 carrying carbapenemase genes. The whole genome sequences were determined by long-read nanopore sequencing. The 12 isolates all contained hybrid plasmids containing both resistance and virulence genes. All carried rmpA/rmpA2 and the aerobactin cluster, with the virulence plasmids of two of three representatives of ST383 carrying blaNDM-5 and seventeen other resistance genes. Representatives of ST48 and ST15 had virulence plasmid-associated genes distributed between two plasmids, both containing antibiotic resistance genes. Representatives of ST101 were remarkable in all sharing virulence plasmids in which iucC and terAWXYZ were missing and iucB and iucD truncated. The combination of resistance and virulence in plasmids of high-risk clones is extremely worrying. Virulence plasmids were often notably consistent within a lineage, even in the absence of epidemiological links, suggesting they are not moving between types. However, there was a common segment containing multiple resistance genes in virulence plasmids of representatives of both STs 48 and 383.

Antibiotics can damage the gut microbiome leading to opportunistic infections and the emergence of antibiotic resistance. Microbiome protection via antibiotic inactivation in the gastrointestinal (GI) tract represents a strategy to limit antibiotic exposure of the colonic microbiota. Proof of concept for this approach was achieved with an orally-administered beta-lactamase enzyme, SYN-004 (ribaxamase), that was demonstrated to degrade ceftriaxone excreted into the GI tract and protect the gut microbiome from antibiotic-mediated dysbiosis. Ribaxamase efficiently degrades penicillin and cephalosporin beta-lactam antibiotics, but is not active against carbapenems. To expand this microbiome protection strategy to include all classes of beta-lactams, three distinct carbapenemases were evaluated for manufacturability, antibiotic degradation spectrum, and stability in human intestinal fluid. E. coli production strains were generated for P2A, a novel metallo-enzyme isolated from B. cereus, New Delhi metallo-beta-lactamase (NDM), and Klebsiella pneumoniae carbapenemase (KPC). While all three enzymes effectively inactivated a broad range of antibiotics, including penicillins, most cephalosporins, and carbapenems in vitro, only P2A retained biological activity when incubated with human chyme. As functional stability in the intestinal tract is a key requirement for an orally-delivered enzyme, P2A was chosen as a potential clinical candidate. An enteric formulation of P2A was developed, called SYN-006, that was inert under high acid conditions, with enzyme dissolution occurring at pH > 5.5. SYN-006 has the potential to expand microbiome protection via antibiotic inactivation to include all classes of beta-lactam antibiotics.

Preventing the spread of multidrug-resistant Gram-negative bacteria (MDRGNB) is a public health priority. However, the definition of MDRGNB applied for planning infection prevention measures such as barrier precautions differs depending on national guidelines. This is particularly relevant in the Dutch–German border region, where patients are transferred between healthcare facilities located in the two different countries, because clinicians and infection control personnel must understand antibiograms indicating MDRGNB from both sides of the border and using both national guidelines. This retrospective study aimed to compare antibiograms of Gram-negative bacteria and classify them using the Dutch and German national standards for MDRGNB definition. A total of 31,787 antibiograms from six Dutch and four German hospitals were classified. Overall, 73.7% were no MDRGNB according to both guidelines. According to the Dutch and German guideline, 7772/31,787 (24.5%) and 4586/31,787 (12.9%) were MDRGNB, respectively (p < 0.0001). Major divergent classifications were observed for extended-spectrum β-lactamase (ESBL) -producing Enterobacteriaceae, non-carbapenemase-producing carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Stenotrophomonas maltophilia. The observed differences show that medical staff must carefully check previous diagnostic findings when patients are transferred across the Dutch–German border, as it cannot be assumed that MDRGNB requiring special hygiene precautions are marked in the transferred antibiograms in accordance with both national guidelines.