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. .

Phenotypic Detection of Enterobacterales Strains Susceptible of Producing OXA-48 Carbapenemase

Background: Nowadays, emergence of Carbapenemase-Producing Enterobacterales (CPE) throughout the world has become a public health problem, especially in countries with limited resources. In recent years, CPE of type OXA-48 (Ambler class D) have been identified in Dakar. The aim of this study was to evaluate the phenotypic detection of OXA-48 CPE using a temocillin disc (30 μg). Methodology: A retrospective study was carried out at Medical Biology Laboratory of Pasteur Institute in Dakar on Ertapenem-Resistant Enterobacterales (ERE) strains isolated from 2015 to 2017. These strains were then tested with a 30 μg temocillin disc. Any strain resistant to temocillin (inhibition diameter Results: Forty-one ERE isolated during the study period were tested, of which 34 (82.9%) were OXA-48 based on phenotypic detection using temocillin disc and confirmed by PCR (100%). OXA-48 CPE strains detected were composed of Klebsiella pneumoniae (n = 14;41.2%), Enterobacter cloacae (n = 8;23.5%), Escherichia coli (n = 7, 20.5%), Citrobacter freundii (n = 3;8.8%), Cronobacter sakazakii (n = 1;3%) and Morganella morganii (n = 1;3%). Conclusion: Temocillin resistance has a good positive predictive value for detecting OXA-48 CPE by phenotypic method, confirmed by PCR. Temocillin is therefore a good marker for detection of OXA-48 CPE except Hafnia alvei.

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.

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.

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.

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.

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.

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.

Dissemination and Genetic Structure of Carbapenemase Encoding Genes (bla_OXA-23and bla_OXA-24) in <i>Acinetobacter baumannii</i>from Southern Texas

Acinetobacter baumannii is one of the most important human pathogens causing a variety of nosocomial infections. Carbapenem antibiotics have been primarily used to treat the A. baumannii infections. However, carbapenem resistant A. baumannii producing carbapenemases causes serious treatment problems worldwide. Outbreaks of carbapenem resistant isolates have reported in some area of the United States, but their dissemination and genetic structure of the carbapenemase encoding genes are currently little known. To understand outbreaks, dissemination, and genetic structure of the carbapenemase encoding genes in Southern Texas, 32 clinical isolates collected from Austin and Houston, TX were characterized. Twenty-eight of 32 isolates were resistant to all tested β-lactam antibiotics including carbapenem (imipenem and meropenem). Three of them carried blaOXA-23 as a part of Tn2008 integrated into a known plasmid (pACICU2) and all others carried blaOXA-24 flanked by XerC/XerD-like recombinase binding sites that were adjoined by DNA sequences originated from multiple plasmids. Genotype analysis revealed that the 25 isolates carrying blaOXA-24 were all identical genotypes same as a representative isolate carrying blaOXA-24 from Chicago, IL but the 3 isolates carrying blaOXA-23 was a distinct genotype as compared with isolates carrying blaOXA-23 from Chicago, IL and Washington, D.C. Each of the blaOXA-23 and blaOXA-24 was transferred to carbapenem susceptible A. baumannii and E. coli with similar minimal inhibitory concentration (MIC) of carbapenem as that of their parental isolates but significantly lower levels of MIC in E. coli. Overall results suggest that a unique strain carrying blaOXA-23 and a similar strain carrying blaOXA-24 as seen in other geographic areas are currently disseminated in Southern Texas.

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.

一株多重耐药大肠杆菌全基因组测序及其耐药性分析

为了解大肠杆菌携带的粘菌素耐药基因并筛选敏感药物,解决多重耐药和动物临床无药可选的困局,采用16S rRNA菌种全基因组测序,PCR筛查mcr-4、mcr-5、blaTEM和AmpC酶耐药基因,应用BLAST和MEGA软件进行生物信息学和系统进化树分析,并对该菌株进行了78种抗生素抗菌药物敏感性测试及4种天然植物提取物(黄藤素、黄连素、黄芩苷和博落回)的抑菌、杀菌效果试验。结果表明,从2021年(1—12月)和2022年(1—6月)猪临床腹泻病例肠道中分离鉴定的145株大肠埃希菌(Escherichia coli,E.coli)中,鉴定出1株同时携带粘菌素耐药基因(mcr-4,mcr-5)和β内酰胺酶blaTEM、AmpC基因的猪源大肠埃希氏菌临床菌株HN2149。78种抗生素药敏试验结果显示,HN2149菌株对头孢吡肟、头孢地嗪、磷霉素、头孢克肟、头孢唑林、头孢哌酮、美洛培南、头孢西丁、头孢呋辛、头孢曲松、头孢噻肟、头孢他啶、替卡西林/克拉维酸、头孢哌酮/舒巴坦、氨曲南、哌拉西林/他唑巴坦、头孢噻肟/克拉维酸、头孢唑肟、头孢美唑、头孢他啶/克拉维酸和头孢他美敏感,对其余57种抗菌药物表现为耐药。4种植物提取物的药敏结果显示,博落回对菌株HN2149的抑菌、杀菌效果较好,其余3种提取物均对该菌株无抑菌和杀菌效果。以上研究结果为猪大肠杆菌病的防控提供参考。

产KPC肺炎克雷伯菌感染治疗的研究进展

近年来全国耐碳青霉烯类肺炎克雷伯菌(CRKP)分离率逐年上升,且由于其多重耐药、病死率高的特点,给临床治疗带来了严峻挑战。CRKP耐药最主要机制为产碳青霉烯酶,在CRKP中常见的碳青霉烯酶类型为Ambler A、B、D类,C类少见。碳青霉烯酶中最常见的是肺炎克雷伯菌碳青霉烯酶(KPC),属于A类。产KPC肺炎克雷伯菌(KPC-KP)在全球范围内广泛扩散,临床有效治疗药物非常有限。本文就KPC-KP感染治疗的研究进展进行总结,以期为临床治疗提供借鉴意义。

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.

建立一种适合本实验室快速检测产KPC酶肺炎克雷伯菌的方法

目的运用基质辅助激光解吸电离飞行时间质谱技术(MALDI-TOF MS)检测产肺炎克雷伯菌碳青霉烯酶(KPC)耐碳青霉烯类肺炎克雷伯菌(CRKP),筛选并验证其特征峰,建立一种适用于本实验室快速检测产KPC酶型CRKP的方法。方法收集临床耐碳青霉烯类肺炎克雷伯菌39株,运用PCR方法检测出其中23株产KPC酶,再采用MALDI-TOF MS技术检测KPC酶阳性CRKP菌株,得到质谱图后用Flex Analyst软件分析筛选出其中特征峰,并进行重复性实验和临床验证评估。结果综合筛选出产KPC酶CRKP最佳特征峰为m/z 6432,特征峰验证实验和重复性实验的特异性和敏感性分别为96.67%和100%。临床验证评估特异性和敏感性分别为92.31%和100%。结论利用MALDI-TOF MS技术可检出产KPC酶CRKP的最佳特征峰为m/z 6432,经实验证实该特征峰特异性和敏感性高,可为临床快速诊断和治疗产KPC酶CRKP提供理论基础。

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.

T6SS阳性CRKP临床感染特征及毒力基因分析

目的分析T6SS阳性耐碳青霉烯类肺炎克雷伯菌(CRKP)临床感染特征,以及其耐药、毒力基因检出率和生物膜形成能力,为临床防控CRKP感染提供参考数据。方法收集2019年1月—2022年12月安徽某三甲医院临床分离的CRKP菌株及患者资料,PCR法检测T6SS基因、毒力基因、耐药基因和分子分型,96孔板结晶紫染色法检测生物膜形成能力。结果共纳入160株CRKP。标本来源以痰(46.9%)和血(26.3%)为主。CRKP菌株呈现多重耐药表型,以携带bla KPC(80.6%)为主,其次为bla NDM(17.5%)。根据是否携带T6SS将CRKP分为T6SS阳性组(129株,80.6%)和T6SS阴性组(31株,19.4%)。T6SS阳性组患者患慢性肺部疾病和心脏疾病比例高于T6SS阴性组(P<0.05),且预后较阴性组差(P<0.05)。T6SS阳性组中,iuc A、mrk D、rmp A2、peg 344、wab G、fim H检出率均高于T6SS阴性组(均P<0.05)。CRKP中以ST11型(68.8%)为主,其中K64-ST11型占比70.9%,K47-ST11型占比25.5%。T6SS阳性组ST11型和K64-ST11型CRKP占比均高于T6SS阴性组(均P<0.05)。T6SS阳性组CRKP生物膜形成能力强于T6SS阴性组(P<0.001)。两组除bla OXA-48基因外,在携带其他碳青霉烯类耐药基因和抗菌药物耐药率方面差异无统计学意义。结论该地区CRKP呈现多重耐药,CRKP菌株T6SS检出率高,T6SS阳性CRKP毒力基因检出率更高,且生物膜形成能力更强。

肝移植术后早期肺炎克雷伯菌感染的临床分析

目的探究肝移植术后早期肺炎克雷伯菌(KP)感染及其对预后的影响。方法回顾性分析171例肝移植受者的资料,根据术后2周细菌培养结果分为无感染组(n=52)和感染组(n=119);感染组86例未检出KP(非KP感染组),33例培养出KP(KP感染组)。比较无感染组和感染组、非KP感染组和KP感染组受者术前、术中及术后资料,分析肝移植术后早期KP感染的危险因素及受者长期生存的影响因素。结果与无感染组比较,感染组术前终末期肝病模型(MELD)评分和总胆红素水平高,手术时间、术后重症监护室(ICU)治疗时间及住院时间长,术中红细胞输注量多,住院费用高,严重并发症发生率高,术后14 d和30 d白细胞计数、中性粒细胞绝对数、中性粒细胞与淋巴细胞比值高,术后14 d淋巴细胞绝对数低,术后30 d血红蛋白低,差异均有统计学意义(均为P<0.05)。与非KP感染组比较,KP感染组MELD评分、总胆红素、天冬氨酸转氨酶(AST)水平高,手术时间、术后ICU治疗时间长,住院费用高,术后90 d病死率高,术后14 d白蛋白低,术后30 d总胆红素高,差异均有统计学意义(均为P<0.05)。33例KP感染受者中,16例对碳青霉烯类抗生素耐药,其中7例在术后90 d内死亡;17例对碳青霉烯类抗生素中介或敏感,4例在术后90 d内死亡。术前MELD评分≥17分、手术时间≥415 min是肝移植术后KP感染的独立危险因素(均为P<0.05)。术后ICU治疗时间≥44 h和KP感染是肝移植长期预后的独立危险因素(均为P<0.05)。结论KP感染是肝移植术后死亡的独立危险因素,术前MELD评分高和手术时间长是肝移植术后早期KP感染的独立危险因素。

2021-2023年某院耐碳青霉烯类肺炎克雷伯菌耐药性和产酶表型分析

目的:分析某三甲医院耐碳青霉烯类肺炎克雷伯菌(Carbapenem-Resistant Klebsiella pneumoniae,CRKP)的临床分布、耐药特点和碳青霉烯酶表型,为制定有效的CRKP控制策略提供参考。方法:回顾性分析2021—2023年某三甲医院住院患者标本中分离的非重复CRKP菌株的分布、常用抗菌药物的耐药性变迁和产酶表型。结果:2021—2023年共检出183株CRKP,主要来源于痰液(65.0%)、尿液(11.5%)和血液(10.4%)。科室分布以ICU(33.3%)为主,其次是呼吸科(24.0%)和神经外科(15.3%)。其中,神经外科构成比呈逐年下降趋势(P<0.05),而康复科构成比则呈逐年上升趋势(P<0.05)。药敏检测结果显示CRKP对β-内酰胺类、氟喹诺酮类和氨基糖苷类药物高度耐药,对替加环素、多黏菌素B和头孢他啶/阿维巴坦较敏感;对左氧氟沙星和环丙沙星的耐药率均呈上升趋势(P<0.05)。大多数CRKP产生碳青霉烯酶,以丝氨酸酶为主,其中产D类丝氨酸酶的(OXA-48)菌株呈逐年下降趋势(P<0.05)。结论:该院临床分离的CRKP菌株主要分布在ICU病房,以肺部感染最为常见。这些菌株对多数常用抗菌药物表现出较高的耐药性,主要与产丝氨酸酶有关。医院需加强重点科室的监控,优化抗菌药物的使用策略,实施精准的防控措施,以有效遏制CRKP的传播。

外科ICU与内科ICU碳青霉烯耐药肺炎克雷伯菌耐药性、碳青霉烯酶基因检测及流行病学研究

目的分析外科ICU和内科ICU碳青霉烯耐药肺炎克雷伯菌(CRKP)的耐药性、常见的碳青霉烯酶基因及菌株之间的亲缘性关系。方法收集2019年1月至2020年12月从某三甲医院外科ICU和内科ICU分离鉴定的CRKP非重复菌株,采用自动仪器法对菌株进行药物敏感性试验;采用mCIM联合eCIM试验进行碳青霉烯酶表型检测,并通过PCR技术联合DNA测序检测碳青霉烯酶基因;利用多位点序列分型(MLST)方法对碳青霉烯酶阳性菌株进行同源性分析。结果共收集15株CRKP菌株,其中外科ICU 8株,内科ICU 7株。CRKP菌株对临床多数抗菌药物表现高耐药性,仅对替加环素表现较高敏感性。mCIM联合eCIM试验并经PCR验证,共有14株CRKP携带碳青霉烯酶基因,其中7株单纯携带KPC⁃2基因,主要来源于内科ICU;5株单纯携带NDM⁃5基因,来源于外科ICU;1株携带KPC⁃2基因合并少见金属酶基因来源于内科ICU;1株携带NDM⁃1基因合并OXA⁃181基因来源于外科ICU。MLST结果显示,医院ICU分离的CRKP菌株为ST11、ST15、ST163个序列型别。内科ICU优势序列为ST11(6/6),全部携带KPC⁃2基因;外科ICU优势序列为ST15(5/8),主要携带NDM⁃5基因。结论医院外科ICU和内科ICU分离的CRKP菌株耐药性高,其携带的耐药基因和优势流行菌株存在差异。应加强多重耐药菌的监测及流行病学研究,及时采取有效治疗方案和防控策略,防止耐药菌株产生和流行播散。