Carbapenemases: A worldwide threat to antimicrobial therapy

Carbapenems are potent β-lactams with activity against extended-spectrum cephalosporinases and β-lactamases. These antibiotics, derived from thienamycn, a carbapenem produced by the environmental bacterium Streptomyces cattleya, were initially used as last-resort treatments forsevere Gram-negative bacterial infections presenting resistance to most β-lactams but have become an empirical option in countries with high prevalence of Extended Spectrum β-lactamase-producing bacterial infections. Imipenem, the first commercially available carbapenem, was approved for clinical use in 1985. Since then, a wide variety of carbapenem-resistant bacteria has appeared, primarily Enterobacteriaceae such as Escherichia coli or Klebsiella pneumoniae(K. pneumoniae), Pseudomonas aeruginosa and Acinetobacter baumannii, presenting different resistance mechanisms. The most relevant mechanism is the production of carbapenem-hydrolyzing β-lactamases, also known as carbapenemases. These enzymes also inactivate all known β-lactams, and some of these enzymes can be acquired through horizontal gene transfer. Moreover, plasmids, transposons and integrons harboring these genes typically carry other resistance determinants, rendering the recipient bacteria resistant to almost all currently used antimicrobials, as is the case for K. pneumoniae carbapenemase- or New Delhi metallo-β-lactamases-type enzymes. The recent advent of these enzymes in the health landscape presents a serious challenge. First, the emergence of carbapenemases limits the currently available treatment options; second, these enzymes pose a risk to patients, as some studies have demonstrated high mortality associated with carbapenemase-producing bacterial infections; and third, these circumstances require an extra cost to sanitary systems, which are particularly cumbersome in developing countries. Therefore, emphasis should be placed on the early detection of these enzymes, the prevention of the spread of carbapenemase-producing bacteria and the development of new drugs resistant to carbapenemase hydrolysis.

Detection of the Production of Klebsiella Pneumoniae Carbapenemase, New Delhi Metallo-Beta-Lactamase and Oxacillinase-48-Type Carbapenemases by Gram-Negative Bacilli in Resource-Limited Setting

Background: The increasing resistance of bacteria to various antibiotics is a worldwide public health issue. Carbapenems that have elicited great hope in treating infections caused by multidrug-resistant germs have seen their efficacy narrowed over time with the emergence of other novel resistance mechanisms, notably the production of Carbapenemases. Methods: A prospective cross-sectional study was conducted from May 2017 to May 2018 in Douala (Cameroon) to detect carbapenemase-producing Gram-negative bacilli. Isolated strains were identified using the Vitek2TM system. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method on agar plates with 20 selected commercially available antibiotic discs. The bacterial strains were tested for the production of three Carbapenemases (OXA-48, NDM, KPC), using an immuno-chromatographic technique, with the “RESIST-3 O.K.N. K-SeT” rapid detection kit. Results: During the study period, 1687 strains of Gram-negative bacilli were isolated in selected laboratories with a total of 200 multi-resistant strains identified (11.9%). Among the multi-resistant strains, E. coli was the species most represented in Enterobacteriaceae (27.5%) followed by K. pneumoniae (15.5%) and the non-fermenting Gram-negative bacilli were predominantly P. aeruginosa (20.5%). These strains mainly came from urine and pus, i.e. 41% and 32% respectively. Thirty-two (16%) strains produced one of the Carbapenemases with a higher frequency at the General Hospital (84%). NDM-type carbapenemase was the most frequently identified (8.5%), OXA-48 type 7.5%, and no KPC production was observed. Among the Enterobacteriaceae 22.9% produced Carbapenemases and only 5.1% of the non-fermenting bacilli produced these enzymes. The isolates strains were completely resistant to all antibiotics except Amikacin and Fosfomycin. The strains producing the NDM-type carbapenemase showed higher rates of resistance to almost all of the antibiotics tested. Conclusion: Multidrug-resistant strains are experiencing an increase in evolution. The apparition of strains producing Carbapenemases prominently, the NDM and OXA-48 favor this increase. The activities of antibiotics with high efficacies on these strains are low.

The Triple Combination of Meropenem,Avibactam,and a Metallo-β-Lactamase Inhibitor Optimizes Antibacterial Coverage Against Different β-Lactamase Producers

This work explores the potential of a triple combination of meropenem(MEM),a novel metallo-blactamase(MBL)inhibitor(indole-2-carboxylate 58(InC58)),and a serine-b-lactamase(SBL)inhibitor(avibactam(AVI))for broad-spectrum activity against carbapenemase-producing bacteria.A diverse panel comprising MBL-and SBL-producing strains was used for susceptibility testing of the triple combination using the agar dilution method.The frequency of resistance(FoR)to MEM combined with InC58 was investigated.Mutants were sequenced and tested for cross resistance,fitness,and the stability of the resistance phenotype.Compared with the double combinations of MEM plus an SBL or MBL inhibitor,the triple combination extended the spectrum of activity to most of the isolates bearing SBLs(oxacillinase-48(OXA-48)and Klebsiella pneumoniae carbapenemase-2(KPC-2))and MBLs(New Delhi metallo-blactamases(NDMs)),although it was not effective against Verona integron-encoded metallo-blactamase(VIM)-carrying Pseudomonas aeruginosa(P.aeruginosa)and OXA-23-carrying Acinetobacter baumannii(A.baumannii).The FoR to MEM plus InC58 ranged from 2.22×10^(-7)to 1.13×10^(-6).The resistance correlated with mutations to ompC and comR,affecting porin C and copper permeability,respectively.The mutants manifested a fitness cost,a decreased level of resistance during passage without antibiotic pressure,and cross resistance to another carbapenem(imipenem)and a b-lactamase inhibitor(taniborbactam).In conclusion,compared with the dual combinations,the triple combination of MEM with InC58 and AVI showed a much wider spectrum of activity against different carbapenemaseproducing bacteria,revealing a new strategy to combat b-lactamase-mediated antimicrobial resistance.

Phenotypic Characterization of Extended Spectrum Beta-Lactamase, Class C Cephalosporinase and Carbapenemase-Producing Klebsiella Species Isolated from Patients Consulted at Four Yaounde-Based Hospitals

Background: Klebsiella spp. are bacteria of medical importance for their role in opportunistic infections which are often difficult to treat because of resistance to one or several antimicrobials. The aim of this study was to determine antimicrobial resistance due to Extended Spectrum Beta-lactamase (ESBL), Class C cephalosporinase (AmpC) and carbapenemase enzymes in Klebsiella spp. isolated from patients consulted at four hospitals. Methodology: The study was cross-sectional and descriptive. A total of 4190 non-repetitive patients specimens from 13 types of clinical specimens were analysed from February to November 2020. Two hundred and twenty-five (225) Klebsiella spp isolates were identified using API 20E and antimicrobial susceptibility testing done according to the Kirby Bauer disc diffusion method. ESBL and AmpC phenotypes were determined by the combination disc method and carbapenemases by double disc synergy method, referenced by EUCAST guidelines for the resistance testing. Results: The frequency of the species was Klebsiella pneumoniae (69%, 155/255), K. oxytoca (14%, 31/255), K. ozaenae (12%, 27/225) and K. rhinoscleromatis (5%, 11/225). Isolates were most resistant to sulphomethoxazole trimethoprim (84%, 189/225), cepaholosporins (80%, 180/225), and least resistant to carbapenems (10.7%, 24/225). Two K. oxytoca and one K. pneumoniae were resistant to all antibiotics tested. Klebsiella pneumoniae had the most multidrug resistant isolates (59.4%, 134/225). Most isolates (83.6%, 188/225) expressed at least one enzyme, while 63.6% (143/225) of the isolates expressed at least two enzymes. Some isolates were ESBL (71.6%, 161/225), carbapenemase (10.7%, 24/225) and AmpC (6.6%, 15/225) producers. Three carbapenemases (Klebsiella pneumoniae carbapenemase-KPC, Metallo-Beta Lactamase-MBL and OXA-48) were detected. Conclusion: These results revealed that resistance of Klebsiella spp. to cephalosporins is high and this may be exacerbated by co-expression of AmpC and carbapenemases aggravating associated patient morbidity and mortality. Monitoring of antimicrobial resistance of local strains is necessary for informed decisions on empirical treatment. .

Dissemination of carbapenem-non-susceptible Acinetobacter baumannii isolates collected from educational hospitals in Qazvin province of Iran

Background:Acinetobacter baumannii(A.baumannii)is known as an opportunistic pathogen related to health-care-associated infection that has a high antibiotic resistance potential,notably against carbapenems that are widely used to combat A.baumannii infections.This study aimed to detect oxacillin-hydrolyzing(OXA)carbapenemases and metallo-β-lactamases(MBL)among carbapenem-resistant A.baumannii isolated strains and to determine their clonal relationship by repetitive extragenic palindromic PCR(rep-PCR).Methods:In the present study,a total of 211 non-repetitive isolates of A.baumannii were collected from Qazvin educational hospitals(2016–2017).The disk diffusion method was used to investigate the antibiotic susceptibility of studied strains,followed by the detection of MBL and OXA-type genes using polymerase chain reaction(PCR)and sequencing methods.The rep-PCR method assessed the clonal relationship of carbapenem-non-susceptible A.baumannii isolates.Result:The obtained results showed that 87.2%and 86.7%of isolates were non-susceptible to imipenem and meropenem.The blaOXA-24(93.5%)was the most frequent gene,followed by the blaOXA-23(4.34%),blaIMP-1(1.63%),and blaVIM-1(0.54%).Meanwhile,blaOXA-58 and blaOXA-143 genes were not found.81.5%and 66.1%of isolates contained ISAba1 upstream of the blaOXA-23 and blaOXA-58 genes,respectively.Rep-PCR results revealed the carbapenem non-susceptible isolates belonged to three distinct clones:A 171(81%),B 34(16.1%),and C 6(2.8%).Conclusions:The results indicated a high prevalence of carbapenem-non-susceptible A.baumannii,with the emergence of the blaOXA-24 gene as the most common gene and the notable prevalence of MBL genes.These results revealed the need for appropriate therapeutic and infection control strategies and monitoring susceptibility patterns for controlling A.baumannii infections.

Identification of blaIMI-mediated carbapenem-resistant Enterobacter from a duck farm in China

Carbapenem-and colistin-resistant Enterobacter has been a clinical and therapy problem in recent years.Here,we report the carbapenem-and colistin-resistant Enterobacter harboring bla_(IMI) isolated from intestinal samples and the environment of a duck farm in China.Four bla_(IMI)-positive Enterobacter isolates were resistant to carbapenem and colistin.Three bla_(IMI) subtypes were detected in different molecular categories of Enterobacter.The detection of the various IMI producers highlights the diversity of carbapenemases in a duck farm.Whole-genome sequencing demonstrated the bla_(IMI) genes were present in chromosomes or plasmids in these strains.The conjugation experiment demonstrated the ability of bla_(IMI)-carrying plasmid to transmit horizontally.The molecular evolution characteristics were examined through comparative genetic analysis.The study demonstrated the presence of chromosomal and plasmid bla_(IMI) and the bla_(IMI)-carrying plasmid exhibits a horizontal transmission between Enterobacter and Escherichia coli C600.The similar genetic content was discovered between two bla_(IMI-16)-positive Enterobacter asburiae.In addition,a bla_(IMI-16)-carrying plasmid is an IncFII(Yp)plasmid,and a substantial amount of mobile genetic elements were identified around bla_(IMI-16).The IS-like elements and IncFII(Yp)plasmid are significant in the propagation of bla_(IMI).Our study provides evidence for the transmission of diverse bla_(IMI) genes in China and supplies additional reference data for bla_(IMI)-positive antimicrobialresistant Enterobacter.Routine surveys of bla_(IMI)-positive Enterobacter from animal-raising environments must be given more focus.

Phenotypic Characterization and Prevalence of Carbapenemase-Producing Acinetobacter baumanii Isolates in Four Health Facilities in Cameroon

Background and Objective: Nowadays, the clinical utility of carbapenems is threatened by the emergence of resistant bacteria, favored by its increasing use. According to the WHO, Acinetobacter baumannii: nosocomial infection agent, tops the list of priority antibiotic-resistant pathogens, considered to be the riskiest for humans. This study sought to determine the prevalence of carbapenemase-producing Acinetobacter baumannii strains in four health facilities in the Center and Littoral regions of Cameroon and the associated risk factors. Materials and Method: An analytical cross-sectional study was conducted over a six-month period from January to June 2022. All suspicious A. baumanii isolates obtained from pathological samples at the bacteriology laboratory of the different health facilities were systematically collected and re-identified. Re-identification and antimicrobial susceptibility Testing (AST) were performed using the VITEK 2 System and the Kirby-Bauer method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Detection and phenotypic characterization of carbapenemases was performed according to adequate standard procedures. Results: A total of 168/226 clinical isolates of Acinetobacter baumannii were confirmed after re-identification, among which 52.69% derived from male patients, 55.09% from participants aged between 10 - 39 years old, and 46.71% from pus samples. A very high resistance rates to all families of antibiotics was noted, except to colistin (10.2%). 40.12% of these isolates produced carbapenemase, represented by 62.69% of class B and 37.31% of class A. Carbapenemase production was observed only at HMR1, Centre region and at Laquintinie hospital, Littoral region with 53.33% and 50% respectively, even if there is no significant difference (P = 0.81). In addition, frequent hospitalisation was significantly associated to the production of carbapenemase among A. baumanii (Adjusted-OR = 16.53, P-value 0.0001). Conclusion: This study highlighted the emergence of carbapenemase-producing Acinetobacter baumannii which is increasingly growing. Continuous drug-resistant monitoring and preventive measures could help to prevent and curb the dissemination of A. baumanii resistance genes, especially in health settings.

Newly Detected Transmission of bla_(KPC-2) by Outer Membrane Vesicles in Klebsiella Pneumoniae

Objective The prevalence of carbapenem-resistant Klebsiella pneumoniae(CR-KP)is a global public health problem.It is mainly caused by the plasmid-carried carbapenemase gene.Outer membrane vesicles(OMVs)contain toxins and other factors involved in various biological processes,includingβ-lactamase and antibiotic-resistance genes.This study aimed to reveal the transmission mechanism of OMV-mediated drug resistance of Klebsiella(K.)pneumoniae.Methods We selected CR-KP producing K.pneumoniae carbapenemase-2(KPC-2)to study whether they can transfer resistance genes through OMVs.The OMVs of CR-KP were obtained by ultracentrifugation,and incubated with carbapenem-sensitive K.pneumoniae for 4 h.Finally,the carbapenem-sensitive K.pneumoniae was tested for the presence of bla_(KPC-2)resistance gene and its sensitivity to carbapenem antibiotics.Results The existence of OMVs was observed by the electron microscopy.The extracted OMVs had bla_(KPC-2)resistance gene.After incubation with OMVs,bla_(KPC-2)resistance gene was detected in sensitive K.pneumoniae,and it became resistant to imipenem and meropenem.Conclusion This study demonstrated that OMVs isolated from KPC-2-producing CR-KP could deliver bla_(KPC-2)to sensitive K.pneumoniae,allowing the bacteria to produce carbapenemase,which may provide a novel target for innovative therapies in combination with conventional antibiotics for treating carbapenem-resistant Enterobacteriaceae.

Phenotypic Characterization of Carbapenemase-Producing Enterobacteriaceae Strains in a Referral Teaching Hospital in Yaoundé, Cameroon

Background: Carbapenemase-producing Enterobacteriaceae (CPE) are an important and increasing threat to global health. They are nowadays more encountered routinely in hospitals and cause high morbidity and mortality due to limited therapeutic alternatives. This study sought to determine the prevalence of CPE in Yaoundé teaching hospital, Cameroon, and the associated risk factors. Materials and Method: To achieve this goal, a descriptive cross-sectional study coupled to an analytical component with consecutive collection of Enterobacteria strains was carried out during a three-month period (from 27th July to 24th October 2018) in the University Teaching Hospital of Yaoundé, Cameroon. The oxidase and biochemical identification tests using a miniaturized Api 20 E system were performed on colonies grown on Eosin Methylene Blue (EMB) medium and subcultured on nutrient agar. Drug susceptibility testing was carried out according to the Antibiogram Committee of the French Society of Microbiology (CA-SFM 2018.V.2.0). The detection of carbapenemase production was performed by the CA-SFM 2018 algorithm for the screening of carbapenemase-producing enterobacteriaceae and its classification by inhibitory synergy tests. Results: Out of the 104 isolates, Escherichia coli (50%) was the most prevalent species, followed by Klebsiella pneumoniae (37.5%) and Citrobacter frendii (12.5%). Drugs susceptibility patterns showed a high resistance to penicillins group (97.4% to amoxicillin), cephalosporins (68.4% to cefotaxim, 58.1% to cefixim, 60.7% to ceftazidim, 57.1% of cefoxitin) and aztreonam (55.7%). However, 11.9% carbapenems related resistance was noticed: 14.4% to imipenem, 13.8% to ertapenem and 7.5% to meropenem. Numerous co-resistance to quinolones (65.8%), fluoroquinolones (49.6%), aminoglycosides (49.6%) and cotrimoxazole (71.8%) were also observed. From 104 isolates, AmpC production represented 23.08% (25/104) and 36.54% (38/104) were ESBL-isolates. The overall prevalence of CPE was 25% (26/104) with K.pneumoniae predominant (61.53%). Besides, Class A and class B carbapenemase were mainly produced with respectively 20% (21/104) and 5% (5/104). Univariate analysis revealed a significant association of carbapenemase production to Klebsiella pneumoniae (p = 0.01), ESBL and AmpC production ((P = 0.01 and P = 0.001 respectively) while that association was only significant to Klebsiella spp (p = 0.04) and AmpC production (p = 0.02) in multivariate analysis. Conclusion: The multi-resistance of Enterobacteriaceae to antibiotics in Cameroon has considerably increased. More attention should be paid to those bacteria to stall antimicrobial resistance spread.

Colistin Resistance Profiles, Molecular Investigation of mcr-1 and mcr-2 Plasmid Genes and Investigation of Carbapenemase Production in Pseudomonas and Acinetobacter Strains

Background and Purpose: The reintroduction of colistin as a last resort treatment against multi-resistant Gram-negative bacilli, is currently challenged by the emergence of colistin-resistant bacteria. The aim of this study was to assess the susceptibility of Pseudomonas and Acinetobacter strains to colistin, to identify carbapenemase production, and to investigate the plasmid genes involved in colistin resistance and carbapenemase production. Methodology: In order to establish the susceptibility profiles of 17 strains of Pseudomonas and Acinetobacter to colistin, their Minimum Inhibitory Concentrations (MICs) were determined using the liquid microdilution method. The possible production of carbapenemases was investigated with the modified Carbapenem Inactivation Method (mCIM). The search for genes encoding carbapenemases (blaOXA, blaIMP, blaCarba) and those responsible for plasmid resistance to colistin (mcr-1 and mcr-2) was performed by conventional PCR. Results and Conclusion: Ninety-four percent (94%) (16/17) of the strains were resistant to colistin. Intraspecies distribution was 50% (8/16), 31% (5/16), 13% (2/16) and 6% (1/16) for Acinetobacter baumannii, Pseudomonas aeruginosa, Pseudomonas luteola, and Pseudomonas fluorescens, respectively. Twenty-nine percent (29%) (6/17) of the strains produced carbapenemases. No mcr-1 and mcr-2 plasmid genes were detected. On the other hand, 17.6% (3/17) of the strains possessed the carbapenemase genes distributed as follows: Carba type (60%), OXA type (40%) and IMP type (0%). The results of this study highlight a high resistance to colistin in strains belonging to the genera Acinetobacter and Pseudomonas, and some of these strains produce carbapenemases.

A Five-year Surveillance of Carbapenemase-producing Klebsiella pneumoniae in a Pediatric Hospital in China Reveals Increased Predominance of NDM-1

Objective To characterize carbapenem （CPM）-non-susceptible Klebsiella pneumoniae （K. pneumoniae） and carbape-nemase produced by these strains isolated from Beijing Children＇s Hospital based on a five-year surveillance. Methods The Minimal Inhibition Concentration values for 15 antibiotics were assessed using the Phonixl00 compact system. PCR amplification and DNA sequencing were used to detect genes encoding carbapenemases. WHONET 5.6 was finally used for resistance analysis. Results In total, 179 strains of CPM-non-susceptible K. pneumoniae were isolated from January, 2010 to December, 2014. The rates of non-susceptible to imipenem and meropenem were 95.0% and 95.6%, respectively. In the 179 strains, 95 （53.1%） strains carried the blalMP gene, and IMP-4 and IMP-8 were detected in 92 （96.8%） and 3 （3.2%） IMP-producing isolates, respectively. 65 （36.3%） strains carried the blaNDM_1 gene. 6 （3.4%） strains carried the blaKpc gene, and KPC-2 were detected in 6 KPC-producing isolates. In addition, New Delhi-Metallo-1 （NDM-1） producing isolates increased from 7.1% to 63.0% in five years and IMP-4 producing isolates decreased from 75.0% to 28.3%. Conclusion High frequencies of multiple resistances to antibiotics were observed in the CPM-non-susceptible K. pneumoniae strains isolated from Beijing Children＇s Hospital. The production of IMP-4 and NDM-1 metallo-13-1actamases appears to be an important mechanism for CPM-non- susceptible in K. pneumoniae.

碳青霉烯类抗生素耐药机制的研究进展

碳青霉烯类抗生素是目前临床上抗菌谱最广、抗菌活性最强的非典型β-内酰胺类抗生素,然而随着碳青霉烯类药物在临床上的广泛应用,细菌对其耐药性不断增加,给临床抗感染治疗带来了新的挑战。为延缓其耐药现象的发展,本文从产生碳青霉烯酶、结合位点改变、外膜孔蛋白缺失或减少、主动外排泵过度表达、细菌生物膜系统等5个方面介绍了碳青霉烯类抗生素的耐药机制,为进行新的药物设计与开发提供依据,同时为临床医师谨慎、合理地应用碳青霉烯类药物提供参考。

Synergistic combination of colistin with imipenem, amikacineor ciprofloxacin against Acinetobacter baumannii andPseudomonas aeruginosa carbapenem-resistant isolated inAnnaba hospital Algeria

Objective:The aim of this study is to detect in vitro the synergetic activity of colistin in combination with imipenem,amikacin or ciprofloxacin,at sub-inhibitory concentrations,against carbapenems-resistant(CR)Acinetobacter baumannii and Pseudomonas aeruginosa strains isolated from various wards in Annaba teaching hospital in eastern Algeria.Materials and Methods:The minimal inhibitory concentrations(MIC)were determined by broth macrodilution(BMD).Carbapenemase encoding genes were screened using polymerase chain reaction(PCR).The activity of colistin in combination with second antibiotic was evaluated by the Checkerboard Technique.Results:39 CR P.aeruginosa and 21 CR A.baumanni strains where collected.The MIC values ranging from(0.25 to 4μg/ml)to colistin,≥16μg/ml for imipenem,≥4μg/ml to amikacin and≥8μg/ml ciprofloxacin.The PCR reveals the presence of the genes blaOXA23(n=12),blaOXA24(n=6),blaNDM1(n=3)in A.baumannii and blaVIM2(n=12)in P.aeruginosa.The combination of colistin with imipenem showed synergistic effect on 57.14%and 46.15%of A.baumannii and P.aeruginosa isolates,respectively.For colistin and amikacin,the synergistic effect is detected in 28.6%of A.baumannii and 30.8%of P.aeruginosa.While colistin and ciprofloxacin showed synergy on 14.29%and 15.38%of A.baumannii and P.aeruginosa isolates,respectively.Conclusion:CR A.baumannii and P.aeruginosa remain the most prevalent infection agents in patients from high-risk wards at Annaba Hospital.Colistin associated with imipenem or with amikacin at sub-inhibitory concentrations gives very encouraging results allowing better management of infections caused by this type of bacteria.

High prevalence of multidrug-resistance in Acinetobacter baumannii and dissemination of carbapenemase-encoding genes bla_(OXA-23-like),bla_(OXA-24-like)and bla_(NDM-1) in Algiers hospitals

Objective:To assess and characterize antibiotic resistance in Acinetobacter baumannii strains recovered from 5 health-care facilities in Algiers.Methods:Antibiotic susceptibility testing was performed by agar diffusion and agar dilution methods,resistance genes were identified by PCR and sequencing,and molecular typing of isolates was carried out by enterobacterial repetitive intergenic consensus-PCR(ERIC-PCR).Results:Among 125 tested isolates,117(93.6% ) were multidrug-resistant.of which 94(75.2% ) were imipenem resistant.The bla_(ADC)and bla_(OXA-51-like) genes were detected in all isolates,in association with ISAba I sequence in 84% and 8% (imipenem resistant) of isolates,respectively.The bla_(OXA-23-like) and bla_(OXA-24-like)carbapenemase genes were delected in 67.02% and 20.21% of imipenem-resistant isolates,respectively.The bla_(OXA-23-like) gene is linked to ISAba1 or ISAba4 elements.The metallo-β-lactamase NDM-1 gene was found in 10(10.6% ) imipenem-resisianl strains from three hospitals,it is linked to ISAba125 clement in nine strains.Extended spectrum β-lactamases production was not detected.Imipenem and cefotaxime resistance phenolypes could not be transferred to Escherichia coli by conjugation.Outer membrane protein CarO gene was not delected in four imipenem-resisianl isolates.The aac(6')-1b.sul1,sul2,tetA and tetB genes were present in 5.31% .36.17% .77.65% .1.06% and 65.92% of strains,respectively.Class 1 integrons were detected in 23.4% strains.KRIC-PCR typing showed a genetic diversity among bla_(OXA-23-like) and bla_(OXA-24-like) positive strains,while clonality was observed among bla_(NDM-1)positives.Conclusions:This study highlighted the high prevalence of imipenem resistance in Acinetobacter baumannii in Algiers hospitals mediated mainly by bla_(OXA-23-like),bla_(OXA-24-like),and bla_(NDM-1) genes.

Genomic and Phenotypic Diversity of Carbapenemase-Producing Enterobacteriaceae Isolates from Bacteremia in China: A Multicenter Epidemiological, Microbiological, and Genetic Study

Carbapenemase-producing Enterobacteriaceae(CPE) isolates are recognized as one of the most severe threats to public health. However, the population structure and genetic characteristics of CPE isolates among bloodstream infections(BSIs) are largely unknown. To address this knowledge gap, in this study,we included patients with clinically significant BSIs due to Enterobacterales isolates, recruited from 26 sentinel hospitals in China(2014–2015). CPE isolates were microbiologically and genomically characterized,including their susceptibility profiles, molecular typing, phylogenetic features, and genetic context analysis of carbapenemase-encoding genes. Of the 2569 BSI Enterobacterales isolates enrolled, 42(1.6%) were carbapenemase-positive. Moreover, among the 2242 investigated isolates, 1111(49.6%) extendedspectrum β-lactamase(ESBL)-producing isolates were identified in Escherichia coli(E. coli), Klebsiella pneumoniae(K. pneumoniae), Proteus mirabilis(P. mirabilis), and Klebsiella oxytoca. Whole genome sequencing analysis showed the clonal spread of K. pneumoniae carbapenemase(KPC)-2-producing K. pneumoniae sequence type(ST) 11 and New Delhi metallo-β-lactamase(NDM)-5-producing E. coli ST167 in our collection. Plasmid analysis revealed that carbapenemase-encoding genes were located on multiple plasmids. A high prevalence of biofilm-encoding type 3 fimbriae clusters and yesiniabactin-associated genes was observed in K. pneumoniae isolates. This work demonstrates the high prevalence of ESBLs and the wide dissemination of CPE among BSI isolates in China, which represent real clinical threats. Moreover, our findings first illustrate a more comprehensive genome scenario of CPE isolates among BSIs. The clonal spread of KPC-2-producing K. pneumoniae ST11 and NDM-5-producing E. coli ST167 needs to be closely monitored.

Characterization of NDM-5-producing Enterobacteriaceae isolates from retail grass carp(Ctenopharyngodon idella) and evidence of blaNDM-5-bearing IncHI2 plasmid transfer between ducks and fish

We aimed to characterize NDM-5-producing Enterobacteriaceae from aquatic products in Guangzhou,China.A total of 196 intestinal samples of grass carp collected in 2019 were screened for carbapenemase genes.Characterization of bla_(NDM-5) positive isolates and plasmids was determined by antimicrobial susceptibility testing,conjugation experiments,Illumina HiSeq,and Nanopore sequencing.One Citrobacter freundii and six Escherichia coli strains recovered from seven intestinal samples were verified as bla_(NDM-5) carriers(3.57%,7/196).The bla_(NDM-5) genes were located on the lncX3(n=5),lncHI2(n=1),or lncHI2-lncF(n=1)plasmids.All bla_(NDM-5)-bearing plasmids were transferred by conjugation at frequencies of~10^(-4)-10^(-6).Based on sequence analysis,the lncHI2 plasmid pHNBYF33-1 was similar to other bla_(NDM-5)-carrying lncHI2 plasmids deposited in GenBank from Guangdong ducks.In all lncHI2 plasmids,bla_(NDM-5)was embedded in a novel transposon,Tn7057(IS3000-△ISAba125-IS5-△ISAba125-bla_(NDM-5)-bleMBL-trpF-tat-△dct-IS26-△umuD-△ISKox3-IS3000),which was identical to the genetic structure surrounding bla_(NDM-5)found in some IncX3 plasmids.The lncHI2-lncF hybrid plasmid pHNTH9F11-1 was formed by homologous recombination of the bla_(NDM-5)-carrying lncHI2 plasmid and a heavy-metal-resistant IncF plasmid through△Tn1721 To the best of our knowledge,this is the first report on the characterization of bla_(NDM-5)-bearing plasmids in fish in China.The lncHI2 plasmid pHNBYF33-1 may be transmitted from ducks,considering the common duck-fish freshwater aquaculture system in Guangdong.Tn7051 is likely responsible for the transfer of bla_(NDM-5) from lncX3 to lncHI2 plasmids in Enterobacteriaceae,resulting in the expansion of transmission vectors of bla_(NDM-5).

Antimicrobial Resistance Patterns and Molecular Characterization of <i>Klebsiella pneumoniae</i>in Clinical Isolates at Mbarara Regional Referral Hospital

Background: Klebsiella pneumoniae is one of the most frequent opportunistic pathogens causing a range of infections and being resistant for beta-lactamases (ESBL) and Carbapenemases. Aim: The aim of the present study was to determine the antimicrobial resistance patterns and molecular characterization establishing the phenotypes and genotypes associated with drug resistance, an antibiogram of genotypically positive isolates for resistance of Klebsiella pneumoniae in clinical isolates at MRRH. Materials and Methods: A laboratory-based descriptive cross-sectional study that was conducted from September 2018 to May 2019 at MRRH. Klebsiella pneumoniae was identified by cultural and biochemical methods. Antibiotic sensitivity test was performed by modified Kirby-Bauer disc diffusion technique. ESBL production in Klebsiella pneumoniae was tested by double-disc synergy test, Carbapenemase production by MHT, Boronic Acid or EDTA test using Meropenem phenotypically and both resistance confirmed genotypically by Multiplex PCR. Results: Out of 1055 clinical isolates, 298 (28.2%) were found positive for Klebsiella.spp, 175 isolates were subcultured among which 22 (12.57%) were K. pneumoniae based on API 20E. Overall Sensitivity patterns of these Klebsiella pneumoniae isolates to Ceftriaxone, (Amoxicillin/Clavulanate), Gentamicin, Cefepime, Ciprofloxacin, Cefoxitin, Nitrofurantoin, Cefuroxime, piperacillin/tazobactam, Meropenem, Ceftazidime and cefotaxime were 72.7%, 63.7%, 54.5%, 45.5%, 31.8%, 31.8%, 27.3%, 27.3%, 22.7%, 22.7%, 18.2%, 9.1%, 9.1% respectively. ESBL producing K. pneumoniae was found at 68.18% (15/22) phenotypically. Genotypically;the ESBL genes were blaCTX-M (100%), blaSHV (80%) and blaTEM (100;47%);8/15 (73.3%) had CTX-M, SHV, TEM, 4/15 (26.67%) CTX-M, TEM, 3/15 (20.00%) CTX-M and SHV. Carbapenemase producing K. pneumoniae was found at 31.82% (7/22) phenotypically;1/7 (14.28%) by MHT, 4/7 (57.14%) Boronic acid test and 2/7 (28.58%) EDTA test. Genotypically;3/4 [(75%) 42.86%] had OXA-48, 1/4 [(25%) 14.28%] OXA-48 and KPC gene, 1/2 [(50%) 14.28%] KPC and VIM, 1/2 [(50%) 14.28%] KPC and KPC gene [(100%) 14.28%]. Conclusion/Recommendations: DDS to be used for ESBL production, MHT, Boronic Acid test and EDTA tests using Meropenem/or Imipenem for Carbapenemase-production routinely.

A comparative study of intestinal Pseudomonas aeruginosa in healthy individuals and ICU inpatients

The human intestinal tract is considered the most important reservoir of the opportunistic pathogens,including Pseudomonas aeruginosa,which is often overlooked but critical due to its antimicrobial resistance and virulence.Public health interventions to control this pathogen require a comprehensive understanding of its epidemiology and genomics.In the current study,we identified P.aeruginosa strains from 2,605 fecal samples collected between 2021 to 2022.Among these samples,574 were from ICU inpatients in Zhejiang province,while 2,031 were obtained from healthy individuals residing in ten different provinces in China.The prevalence of P.aerugi-nosa intestinal carriage was found to be higher in ICU inpatients(10.28%,95%CI:7.79%-12.76%)than that in healthy individuals(3.99%,81/2,031,95%CI:3.14%-4.84%).Similarly,the prevalence of carbapenem-resistant P.aeruginosa(CRPA)was higher in ICU inpatients(32.2%)compared to healthy individuals(7.41%).The population structure analy-sis of our isolates revealed a predominantly non-clonal distribution,with 41 distinct sequence types identified among 59 P.aeruginosa isolates from ICU inpatients and 38 different STs among 81 P.aeruginosa isolates from healthy individuals.These findings suggest that the individual acquisition of P.aeruginosa is more frequent than patient-to-patient transmission,as evidenced by the polyclonal population structure.Antimicrobial susceptibility testing and genome analysis indicated that P.aeruginosa strains from ICU inpatients exhibited significantly higher resistance rates to most antimicrobials and harbored a greater number of acquired resistance genes compared to strains from healthy individuals.Notably,in ICU inpatients,we identified three isolates of ST463,all of which shared the conserved Tn3-TnpR-ISKpn8-blaKPC-ISKpn6 genetic context.Additionally,five isolates carrying the qacE gene were also identified,these findings suggest that small-scale transmission events may still occur within the ICU setting,posing significant challenges for clinical management.With regard to virulence factors,we observed similar profiles between the two groups,except for phzA2,phzB2,and pilA,which were statistically higher in isolates from healthy individuals.This may be because the accumulating resistance mutations in ICU-derived P.aeruginosa are linked to a decrease in virulence.

Emergence of Klebsiella pneumoniae carbapenemase-producing Proteus mirabilis in Hangzhou, China

Background Carbapenems are used to treat severe infections caused by multi-drug-resistant organisms, however, the emergence of carbapenem-resistant bacterial isolates is becoming an increasing therapeutic challenge. Since the first Klebsiella （K.） pneumoniae carbapenemase （KPC）-producing K. pneumoniae was reported in 2001, KPC-producing isolates have been found increasingly, specially in Enterobacteriaceae. The aim of this study was to characterize the mechanisms of a carbapenem-resistant Proteus （P.） mirabilis. Methods A carbapenem-resistant P. mirabilis isolate was recovered from pleural drainage fluid of a patient admitted to surgical intensive care unit. Antimicrobial susceptibility testing of the isolate was performed by disk diffusion according to Clinical and Laboratory Standards Institute guidelines, and subsequent minimal inhibitory concentrations were determined with the E-test. Amplification of the blaKPC gene generated a positive band and the PCR products were sequenced subsequently. The plasmid of the isolate was extracted and was successfully transformed into Escherichia （E.） coli DH5a. Results The P. mirabilis isolate was resistant to all detected antimicrobial agents except tigecycline. KPC-2 was confirmed by DNA sequence analysis. The transformant E. coil was resistant to carbapenems. Further study demonstrated that upstream and downstream regions of b/aKPC-2 were identical to that observed in K. pneumoniae submitted to GenBank from China in 2007. Conclusion Carbapenem resistance in the P. mirabilis isolate in this study is mainly due to production of KPC-2.

Genomic Epidemiology of Carbapenemaseproducing Klebsiella pneumoniae in China

The rapid spread of carbapenemase-producing Klebsiella pneumoniae(cpKP)poses serious threats to public health;however,the underlying genetic basis for its dissemination is still unknown.We conducted a comprehensive genomic epidemiology analysis on 420 cpKP isolates collected from 70 hospitals in 24 provinces/autonomous regions/municipalities of China during 2009–2017 by short-/long-read sequencing.The results showed that most cpKP isolates were categorized into clonal group 258(CG258),in which ST11 was the dominant clone.Phylogenetic analysis revealed three major clades including the top one of Clade 3 for CG258 cpKP isolates.Additionally,carbapenemase gene analysis indicated that blaKPC was dominant in the cpKP isolates,and most blaKPC genes were located in five major incompatibility(Inc)groups of blaKPC-harboring plasmids.Importantly,three advantageous combinations of host–blaKPC-carrying plasmid(Clade 3.1+3.2–IncFIIpHN7A8,Clade 3.1+3.2–IncFIIpHN7A8:IncR,and Clade 3.3–IncFIIpHN7A8:IncpA1763-KPC)were identified to confer cpKP isolates the advantages in both genotypes(strong correlation/coevolution)and phenotypes(resistance/growth/competition)to facilitate the nationwide spread of ST11/CG258 cpKP.Intriguingly,Bayesian skyline analysis illustrated that the three advantageous combinations might be directly associated with the strong population expansion during 2007–2008 and subsequent maintenance of the population of ST11/CG258 cpKP after 2008.We then examined drug resistance profiles of these cpKP isolates and proposed combination treatment regimens for CG258/non-CG258 cpKP infections.Thus,the findings of our systematical analysis shed light on the molecular epidemiology and genetic basis for the dissemination of ST11/CG258 cpKP in China,and much emphasis should be given to the close monitoring of advantageous cpKP–plasmid combinations.