Role of outer membrane protein OprD and penicillin-binding proteins in resistance of Pseudomonas aeruginosa to imipenem and meropenem

The main mechanism of imipenem resistance in Pseudomonas aeruginosa is via downregulation of the gene for OprD porin. In a previous study, it was shown that the level of resistance did not parallel with the degree of downregulation of the porin gene, thus arguing for the existence of other resistance mechanisms. Penicillin-binding protein (PBP) 2 and PBP3 are involved in carbapenem resistance in Escherichia coli. The genes for PBPs were sequenced in three imipenem-resistant clinical strains and these strains were conjugated with two susceptible P. aeruginosa PA0 strains, selecting for auxotrophic markers. In all the clinical and resistant isolates there was no obvious elevation of AmpC cephalosporinase. The active sites of PBP1b (ponB), PBP2 (pbpA), PBP3 (pbpB) and PBP6 (dacC) had no mutations in any of the examined strains. Production of oprD mRNA was significantly lower in clinical strains and transconjugants after selection for the proB marker (PA4565 at 5113 kb). The clinical strains had alterations in OprD that were not found in transconjugants. Our findings suggest that PBPs do not play a role in imipenem resistance in the clinical strains examined here, but that a regulatory gene for oprD contributing to carbapenem resistance is located close to the proB gene.     

Detection of the icaADBC gene cluster and biofilm formation in Staphylococcus epidermidis isolates from catheter-related urinary tract infections

Biofilm production in Staphylococcus epidermidis is an important virulence factor that is mediated by the expression of the icaADBC operon. In this study 41 S. epidermidis isolates obtained from catheter-related urinary tract infections were analyzed for the presence of the icaADBC operon and biofilm formation. Eighteen of 41 isolates (44%) were shown to carry ica-specific DNA, but only 11 isolates (27%) produced biofilms spontaneously under normal growth conditions. Upon induction by external stress or antibiotics, biofilm formation could be stimulated in five of seven ica-positive, biofilm-negative isolates, indicating that the icaADBC expression was down-regulated in these strains. Genetic analyses of the ica gene clusters of the remaining two ica-positive, biofilm-negative strains revealed a spontaneous icaC::IS256 insertion in one strain. Insertion of the element caused a target site duplication of seven base pairs and a biofilm-negative phenotype. After repeated passages the insertion mutant was able to revert to a biofilm-forming phenotype which was due to the precise excision of IS256 from the icaC gene. The data show that icaC::IS256 integrations occur during S. epidermidis polymer-related infections and the results highlight the biological relevance of the IS256-mediated phase variation of biofilm production in S. epidermidis during an infection.     

In vitro activity of doripenem against Acinetobacter baumannii clinical isolates

Doripenem is a carbapenem with activity against Gram-positive and Gram-negative pathogens. This study evaluated the in vitro activity of doripenem against a collection of 87 Acinetobacter baumannii clinical isolates, showing that the activity of doripenem was superior to imipenem and meropenem for strains carrying the bla(OXA-58) gene. A. baumannii clinical isolates expressing the bla(OXA-24) gene were resistant to doripenem, imipenem and meropenem. However, in clinical isolates expressing the bla(OXA-58) gene, the percentage of isolates with a doripenem minimum inhibitory concentration >8microg/mL was much lower than that of imipenem and meropenem. This study shows that the activity of doripenem was superior to imipenem and meropenem for strains carrying the bla(OXA-58) gene.     

High prevalence of OXA-143 and alteration of outer membrane proteins in carbapenem-resistant Acinetobacter spp. isolates in Brazil

Carbapenem resistance amongst Acinetobacter spp. has been increasing in the last decade. This study evaluated the outer membrane protein (OMP) profile and production of carbapenemases in 50 carbapenem-resistant Acinetobacter spp. isolates from bloodstream infections. Isolates were identified by API20NE. Minimum inhibitory concentrations (MICs) for carbapenems were determined by broth microdilution. Carbapenemases were studied by phenotypic tests, detection of their encoding gene by polymerase chain reaction (PCR) amplification, and imipenem hydrolysis. Nucleotide sequencing confirming the enzyme gene type was performed using MegaBACE 1000. The presence of OMPs was studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and PCR. Molecular typing was performed using pulsed-field gel electrophoresis (PFGE). All isolates were resistant to carbapenems. Moreover, 98% of the isolates were positive for the gene encoding the enzyme OXA-51-like, 18% were positive for OXA-23-like (only one isolate did not show the presence of the insertion sequence ISAba1 adjacent to this gene) and 76% were positive for OXA-143 enzyme. Five isolates (10%) showed the presence of the IMP-1 gene. Imipenem hydrolysing activity was detected in only three strains containing carbapenemase genes, comprising two isolates containing the bla(IMP) gene and one containing the bla(OXA-51/OXA-23-like) gene. The OMP of 43 kDa was altered in 17 of 25 strains studied, and this alteration was associated with a high meropenem MIC (256 μg/mL) in 5 of 7 strains without 43 kDa OMP. On the other hand, decreased OMP 33-36 kDa was found in five strains. The high prevalence of OXA-143 and alteration of OMPs might have been associated with a high level of carbapenem resistance.     

Occurrence of PER-1 producing clinical isolates of Pseudomonas aeruginosa in Japan and their susceptibility to doripenem

The acquisition of resistance by extended-spectrum beta-lactamases (ESBL) has been reported primarily for Enterobacteriaceae, but there are few reports on the isolation of ESBL-producing Pseudomonas aeruginosa. PER-1-type ESBL producing P aeruginosa has been found in various regions around the world but there are no reports of clinical isolates in Japan. During our susceptibility surveillance studies over a 10 year period, we found four clinical isolates resistant to ceftazidime due to production of PER-1. They were resistant to ceftazidime but susceptible in the presence of clavulanic acid, a class A beta-lactamase inhibitor. The strains had the ability to hydrolyze ceftazidime. They also had the gene for PER-1-type ESBL. This is the first report of the isolation of PER-1 producing strains in Japan. These four strains were resistant to ceftazidime, cefepime and aztreonam with MICs of 64 microg/ml or more, but were more susceptible to carbapenem antibiotics. In particular, doripenem, which is a novel carbapenem antibiotic, showed good antibacterial activity with a MIC of 2 or 4 microg/ml, which was more potent than meropenem and imipenem. Doripenem also showed good therapeutic efficacy against a systemic infection of mice with a PER-1 producing strain, and was also more potent in vivo than imipenem or meropenem.     

Ertapenem resistance among Klebsiella and Enterobacter submitted in the UK to a reference laboratory

The emergence of carbapenem resistance in Enterobacteriaceae represents a major public health concern. We investigated ertapenem-resistant clinical isolates of Klebsiella spp. and Enterobacter spp. referred to the UK's national reference laboratory for antibiotic resistance. Minimum inhibitory concentrations (MICs) were determined and interpreted according to British Society for Antimicrobial Chemotherapy guidelines. Genes for carbapenemases and CTX-M extended-spectrum beta-lactamases (ESBLs) were sought by polymerase chain reaction, and imipenem hydrolysis was determined by spectrophotometry with crude extracts. From June 2004 to April 2006, 95 Klebsiella spp. and 76 Enterobacter spp. isolates resistant to ertapenem (MICs >2mg/L) were received, 40% of which (38 Klebsiella spp. and 30 Enterobacter spp.) were highly resistant to ertapenem (MICs >16mg/L). Imipenem and meropenem were active (geometric mean MICs <2mg/L) against most isolates with low-level ertapenem resistance but were less active against highly ertapenem-resistant isolates. Only one ertapenem-resistant isolate produced a defined carbapenemase, a Klebsiella pneumoniae with IMP-1 enzyme; one Enterobacter sp. also hydrolysed imipenem, but its carbapenemase remains to be identified. Geometric mean MICs of ertapenem for the collection were reduced five-fold by clavulanic acid for Klebsiella spp. compared with eight-fold by cloxacillin for Enterobacter spp. This study highlights the fact that ertapenem resistance is being detected in Klebsiella spp. and Enterobacter spp. in the UK, but that it is rarely mediated by true carbapenemases. Rather, it probably results from combinations of a beta-lactamase - often a CTX-M ESBL in Klebsiella spp. or an AmpC enzyme in Enterobacter spp. - plus impermeability and/or increased efflux. Imipenem and meropenem often remain moderately active against isolates with low-level ertapenem resistance.     

Faropenem resistance causes in vitro cross-resistance to carbapenems in ESBL-producing Escherichia coli

ObjectiveFaropenem is an oral penem drug with activity against Gram-positive and Gram-negative bacteria, including CTX-M-15-type extended spectrum beta-lactamase (ESBL)-producing Enterobacteriales and anaerobic bacteria. As there are structural similarities, there is concern for the development of carbapenem cross-resistance; however, there are no studies confirming this. This study examined whether in vitro development of faropenem resistance in Escherichia coli isolates would result in cross-resistance to carbapenems.MethodsFour well-characterized E. coli isolates from the US Centers for Disease Control and Prevention antibiotic resistance isolate bank were utilized. Three isolates (NSF1, NSF2 and NSF3) are ESBL producers (CTX-M-15) and one (NSF4) is pan-susceptible. Faropenem minimum inhibitory concentrations (MICs) were determined and resistance was induced by serial passaging in increasing concentrations of faropenem. Susceptibility to carbapenems was determined and whole-genome sequencing (WGS) was performed to identify the underlying genetic mechanism leading to carbapenem resistance.ResultsFaropenem MIC increased from 1 mg/L to 64 mg/L within 10 days for NSF2 and NSF4 isolates, and from 2 mg/L to 64 mg/L within 7 days for NSF1 and NSF3 isolates. Reduced carbapenem susceptibility (ertapenem MIC ≥8 mg/L, doripenem/meropenem ≥2 mg/L and imipenem ≥1 mg/L) developed among three CTX-M-15-producing isolates that were faropenem-resistant, but not in NSF4 isolate that lacked ESBL enzyme. WGS analysis revealed non-synonymous changes in the ompC gene among three CTX-M-15-producing isolates, and a single nucleotide polymorphism (SNP) in the envZ gene in NSF4 isolate.ConclusionInduced resistance to faropenem causes cross-resistance to carbapenems among E. coli isolates containing CTX-M-15-type ESBL enzymes.     

In vitro activity of ceftiofur tested against clinical isolates of Escherichia coli and Klebsiella pneumoniae including extended spectrum β-lactamase producing strains

In vitro activity of ceftiofur, a cephalosporin used in veterinary practice was compared using ceftriaxone-resistant (producing extended spectrum β-lactamase (ESBL)) and -susceptible clinical isolates of Esherichia coli and Klebsiella pneumoniae. The ceftriaxone-susceptible isolates exhibited a lower range of ceftiofur MICs (MIC₅₀, 0.5 mg/l, MIC₉₀ 1.0 mg/l). Those isolates known to produce an ESBL were also resistant to ceftiofur (MIC₅₀, ≥ 32 mg/l). The latter isolates were also less susceptible to other comparator drugs (cefquinome, gentamicin and trimethoprim/sulphamethoxazole) in contrast to the ceftriaxone-susceptible strains. The clinical isolates showed high correlation between ceftriaxone and ceftiofur MICs (y=2.6+0.89x, r=0.95). Using the current ceftiofur susceptible breakpoint (≤2 mg/l) used for veterinary practice (respiratory tract pathogens), the ESBL-producing strains of E. coli and K. pneumoniae could be accurately separated from susceptible strains. This ceftiofur breakpoint MIC corresponds to the National Committee for Clinical Laboratory Standards ESBL screening concentration for ceftriaxone set at ≤1 mg/l=negative for ESBL production. Ceftiofur was also observed to be very active in vitro against ampicillin-resistant, non-ESBL producing enteric isolates. This new cephem appears to be very potent against the tested Enterobacteriaceae and of potential wide clinical veterinary utility.     

Emergence of imipenem resistance in clinical Escherichia coli during therapy

The molecular epidemiology and the mechanisms of resistance of Escherichia coli isolated from two patients infected by imipenem-resistant strains are reported in this study. From one patient, three closely related consecutive isolates of E. coli were recovered; the first was carbapenem-susceptible but acquired imipenem resistance after treatment with ertapenem, and the third isolate was again imipenem-susceptible. An additional imipenem-resistant isolate was recovered from another patient who received imipenem. The genetic relatedness of the E. coli isolates was determined by pulsed-field gel electrophoresis (PFGE) after digestion with XbaI. Standard polymerase chain reaction (PCR) conditions were used to amplify several beta-lactamase genes coding for carbapenemases, extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated AmpC; the E. coli ampC gene promoter was also amplified and sequenced. Primers OmpF-F/OmpF-R and OmpC-F/OmpC-R were used to amplify the ompF and ompC genes. The outer membrane protein (OMP) profiles were studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Imipenem-resistant E. coli isolates did not produce carbapenemases but lacked the two major OMPs OmpF and OmpC and had ampC promoter mutations; in addition, one of the imipenem-resistant isolates produced the CMY-2 cephalosporinase, whilst the other produced the new CTX-M-67 ESBL. Carbapenem resistance in this study was associated with lack of expression of OmpF and OmpC porins. Additional mechanisms of beta-lactam resistance, such as plasmid-mediated AmpC and ESBL production, were also found. Development of carbapenem resistance in a CTX-M-67-producing E. coli is first described in this study.     

舒巴坦联合9种抗菌药物对耐碳青霉烯类鲍曼不动杆菌体外抗菌活性研究

目的：评价舒巴坦与9种临床常用抗菌药物联合用药对耐碳青霉烯类鲍曼不动杆菌（CRAB）的体外抗菌效应。方法：从临床分离得到24株CRAB。采取微量肉汤稀释法测定10种抗菌药物单一用药对CRAB的最低抑菌浓度（MIC）。采用棋盘法设计,微量肉汤稀释法测定舒巴坦分别联合9种抗菌药物对CRAB的MIC,并计算部分抑菌浓度指数（FIC）判定联合效应。结果：9种抗菌药物联用舒巴坦后的MIC均比单用时降低;联用后替加环素、多西环素、环丙沙星、左氧氟沙星的MIC₅₀均低于对应单药敏感折点,替加环素的MIC₉₀达到单药的敏感折点。舒巴坦与9组联合用药均未出现拮抗作用;舒巴坦联合阿米卡星的协同作用最强,其次为美罗培南、帕尼培南、亚胺培南、多西环素、替加环素。结论：与单药相比,9种药物联用舒巴坦对CRAB的体外抑菌作用均有增强效果。9种抗菌药物联合舒巴坦有效地降低MIC值,其中多西环素、替加环素、环丙沙星和左氧氟沙星,可使其MIC值降至敏感范围内。     

In vivo development of carbapenem resistance in clinical isolates of Enterobacter aerogenes producing multiple beta-lactamases

Four clinical strains of extended-spectrum beta-lactamase- and AmpC-producing Enterobacter aerogenes were isolated successively from a liver transplantation patient. Isolates C(1) and C(2) were isolated prior to carbapenem therapy, whilst isolates C(3) and C(4) were recovered after 40 days of carbapenem therapy. The homology of these strains was analysed by pulsed-field gel electrophoresis (PFGE). beta-Lactamases were analysed by isoelectric focusing, polymerase chain reaction (PCR) and sequencing. Outer membrane proteins were analysed by PCR, sequencing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot. Disruption of OmpE36 in C(1) in vitro was also performed by homologous gene recombination. The isolates demonstrated an indistinguishable PFGE pattern. Molecular characterisation revealed that, in addition to the pre-existing multiple beta-lactamases (DHA-1, TEM-1, SHV-5, CTX-M-3 and CTX-M-14) found in C(1) and C(2), isolates C(3) and C(4) failed to express OmpE36 owing to insertional inactivation by an IS903-like insertion sequence. Other resistance mechanisms, such as production of carbapenem-hydrolysing enzymes or expression of chromosomal efflux, were apparently not involved. Completely replacing OmpE36 by the kanamycin resistance gene (kan) resulted in a significant increase in carbapenem minimum inhibitory concentrations of an ompE36 mutant. Thus, C(3) and C(4) were apparently derived from the previously imipenem-susceptible isolates C(1) and C(2). Following carbapenem exposure, depletion of OmpE36 expression resulted in the collateral effect of carbapenem resistance.     

Comparative in vitro activity of carbapenems against major Gram-negative pathogens: results of Asia-Pacific surveillance from the COMPACT II study

Resistance rates amongst Gram-negative pathogens are increasing in the Asia-Pacific region. The Comparative Activity of Carbapenem Testing (COMPACT) II study surveyed the carbapenem susceptibility and minimum inhibitory concentrations (MICs) of doripenem, imipenem and meropenem against 1260 major Gram-negative pathogens isolated from hospitalised patients at 20 centres in five Asia-Pacific countries (New Zealand, the Philippines, Singapore, Thailand and Vietnam) during 2010. Pseudomonas aeruginosa (n=625), Enterobacteriaceae (n=500), and other Gram-negative pathogens including Acinetobacter baumannii (n=135) were collected from patients with bloodstream infection (32.2%), nosocomial pneumonia including ventilator-associated pneumonia (58.1%), and complicated intra-abdominal infection (9.7%), with 36.7% being isolated from patients in an Intensive Care Unit. As high as 29.8% of P. aeruginosa and 73.0% of A. baumannii isolates were not susceptible to at least a carbapenem, whereas the majority of Enterobacteriaceae (97.2%) were susceptible to all carbapenems. Respective MIC(50)/MIC(90) values (MICs for 50% and 90% of the organisms, respectively) of doripenem, imipenem and meropenem were: 0.38/8, 1.5/32 and 0.38/16 mg/L for P. aeruginosa; 0.023/0.094, 0.25/0.5 and 0.032/0.094 mg/L for Enterobacteriaceae; and 32/64, 32/128 and 32/64 mg/L for A. baumannii. Doripenem and meropenem had comparable activity against P. aeruginosa, both being more active than imipenem. All carbapenems were highly potent against Enterobacteriaceae, although imipenem demonstrated higher MIC values than doripenem and meropenem. The three carbapenems showed less activity against A. baumannii. The high prevalence of carbapenem resistance amongst important nosocomial pathogens (P. aeruginosa and A. baumannii) warrants rigorous infection control measures and appropriate antimicrobial use in the Asia-Pacific region.     

How carbapenem-resistant Acinetobacter spp. established in a newly constructed hospital

Annually increasing rates of carbapenem-resistant Acinetobacter spp. were observed in a Taiwan hospital since its establishment in November 1998 to March 2005. Increasing consumption of carbapenems was also noticed. Carbapenem-resistant Acinetobacter spp. from 33 patients carried a class 1 integron. Twenty-eight isolates were Acinetobacter baumannii harbouring both ISAba1 and an OXA-51-like gene. Twenty-four of the 28 A. baumannii isolates had ISAba1 upstream of the OXA-51-like gene. Four A. baumannii isolates harboured the OXA-24-like gene and one isolate had the VIM-11 gene. Regarding the five non-baumannii Acinetobacter spp., three Acinetobacter genomic species 3 isolates and one Acinetobacter radioresistens isolate had both IMP-1 and OXA-58-like genes. One A. radioresistens isolate had an OXA-23-like gene. One major clone of A. baumannii (25/28; 89.3%) was identified by ribotyping. Three ribotypes were identified as being brought into the hospital by patient transfer from other hospitals. In conclusion, an insidious clonal dissemination with various resistance mechanisms contributed to the spread of carbapenem-resistant Acinetobacter spp. in a hospital setting, with increasing usage of carbapenems as the possible selection pressure. Notification of carbapenem-resistant Acinetobacter spp. infection when patients are transferred between hospitals is important to control the spread of carbapenem resistance.     

Distribution of tetracycline resistance genes tet(M), tet(O), tet(L) and tet(K) in blood isolates of viridans group streptococci harbouring erm(B) and mef(A) genes. Susceptibility to quinupristin/dalfopristin and linezolid

Susceptibility to erythromycin, tetracycline, clindamycin, quinupristin/dalfopristin and linezolid was investigated using 111 consecutive non-duplicate blood culture isolates of viridans-group streptococci (VGS). The erm(B) and mef(A) genes were detected, either alone or in combination, in the 47 (42%) erythromycin-resistant strains. The tet(M) gene alone was predominant (78%) in the 36 (35%) tetracycline-resistant isolates. Two isolates carried the tet(O) gene alone and two others the tet(L) associated with tet(O) or tet(M). The association between erythromycin and tetracycline resistance was common and the erm(B) and tet(M) determinants seem to be associated in our VGS. We found three isolates resistant to quinupristin/dalfopristin, all of them were erythromycin and tetracycline-resistant. For all isolates tested, linezolid MICs were ≤2 mg/l.     

某院肠杆菌科细菌耐药性与抗菌药物使用强度时间相关性分析

目的　探讨肠杆菌科细菌耐药性与抗菌药物使用强度时间相关性,为优化抗菌药物临床选用提供依据。方法　采用回顾性调查方法,统计连云港市第一人民医院2016年1月至2021年12月期间住院患者各类标本中检出肺炎克雷伯菌、大肠埃希菌对哌拉西林/他唑巴坦、美罗培南以及亚胺培南耐药率和同期β‐内酰胺酶复合制剂、碳青霉烯类抗菌药物使用强度。采用线性回归分析抗菌药物季度使用强度和细菌季度耐药率变化趋势,运用互相关函数分析抗菌药物季度使用强度与细菌季度耐药率之间时间相关性。结果　6年期间肺炎克雷伯菌对哌拉西林/他唑巴坦（P<0.001）、美罗培南（P=0.001）耐药率均呈上升趋势,大肠埃希菌对哌拉西林/他唑巴坦耐药率逐渐下降（P<0.001）。研究期间碳青霉烯类抗菌药物使用强度呈上升趋势（P=0.008）,β-内酰胺酶复合制剂类抗菌药物使用强度呈下降趋势（P=0.032）。肺炎克雷伯菌对哌拉西林/他唑巴坦耐药率与碳青霉烯类抗菌药物使用强度高度同步（r=0.812,Lag=0,P=0.022）,对美罗培南（r=0.704,Lag=1,P=0.037）和亚胺培南（r=0.885,Lag=1,P=0.005）耐药率与碳青霉烯类抗菌药物使用强度均呈显著正相关。β-内酰胺酶复合制剂类抗菌药物使用强度与大肠埃希菌（r=0.587,Lag=3,P=0.045）和肺炎克雷伯菌（r=0.531,Lag=2,P=0.039）对哌拉西林/他唑巴坦耐药率均呈正相关。结论　医院肠杆菌科细菌耐药率与抗菌药物使用强度存在一定时间相关性,临床应优化抗菌药物合理选用,遏制肠杆菌科细菌耐药性进展。     

Emergence of carbapenem resistance in Bacteroides fragilis in China

The antimicrobial resistance crisis makes it critically important for laboratories to closely monitor trends and mechanisms of emerging antimicrobial resistance in clinical isolates. Bacteroides fragilis is an anaerobic pathogen that causes several serious infections and is increasingly resistant to antimicrobial agents. However, data from China regarding antimicrobial resistance in B. fragilis are limited. In this work, the mechanism underlying carbapenem resistance in 44 B. fragilis isolates collected from a Chinese hospital was investigated. Antimicrobial susceptibility testing for 13 antimicrobial agents was performed by the agar dilution method, and the contribution of efflux pumps to carbapenem resistance was analysed. Genetic relatedness of the isolates was determined by PFGE. Outer membrane porins were analysed in isolates with reduced carbapenem susceptibility. Potential carbapenemase-encoding genes were identified, and the location and environment of the cfiA gene was analysed. Among the 44 isolates, 18.2%, 29.5%, 22.7%, 100%, 100%, 29.5%, 15.9%, 81.8%, 88.6% and 47.7% were resistant to imipenem, meropenem, ertapenem, penicillin, ampicillin, amoxicillin/clavulanic acid, piperacillin/tazobactam, clindamycin, tetracycline and moxifloxacin, respectively. None of the isolates were resistant to metronidazole, cefoxitin or chloramphenicol. A chromosomally located gene (cfiA) encoding a metallo-β-lactamase was identified in 16 isolates (36.4%). A conserved insertion sequence of IS1187 or IS613 was upstream of cfiA in eight isolates with high-level carbapenem resistance. The insertion sequences were associated with increased carbapenem resistance in B. fragilis by upregulating the expression of cfiA as shown by RT-qPCR. This is the first study to describe a mechanism of carbapenem resistance in B. fragilis in mainland China.     

Carbapenem-resistant Escherichia coli and Klebsiella pneumoniae isolates from Turkey with OXA-48-like carbapenemases and outer membrane protein loss

Treatment options are limited in infections caused by extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, with carbapenems generally preferred. Disturbingly, however, carbapenem-resistant strains are emerging worldwide. Here we report two clinical isolates, one Escherichia coli and one Klebsiella pneumoniae, each with high-level carbapenem resistance (imipenem minimum inhibitory concentration of 32 microg/mL). They were isolated following imipenem therapy from two hospital patients who had received imipenem therapy in different regions of Turkey. Both isolates produced OXA-48-like carbapenemases, enzymes so far reported only from Turkey. Both isolates also had group 1 CTX-M-type ESBLs and had lost major outer membrane proteins. OXA-48-like carbapenemases appear to be scattered in Turkey and surveillance to determine their prevalence is warranted.     

我院鲍曼不动杆菌携带blaOXA-23基因及其耐药机制研究

目的研究我院分离鲍曼不动杆菌携带bla_OXA-23基因的携带情况及其诱导细菌耐药机制。方法收集江阴市青阳医院2019年1月至2019年12月间临床分离鲍曼不动杆菌,经VITEK-2全自动微生物鉴定及药敏分析系统鉴定耐亚胺培南菌株100株,敏感菌株100株。通过多重PCR法检测上述菌株中bla_OXA-23、bla_OXA-24、blaOXA-51和bla_OXA-58基因携带情况。通过PCR法扩增亚胺培南耐药的鲍曼不动杆菌bla_OXA-23的ORF序列,构建过表达载体bla_OXA-23/pYMAb3并电转化鲍曼不动杆菌(ATCC 17978),以硫酸卡那霉素和美罗培南共同筛选过表达bla_OXA-23基因的菌株。采用稀释法检测转化bla_OXA-23/pYMAb3载体和pYMAb3空载体的菌株对亚胺培南的MIC。结果我院耐亚胺培南的鲍曼不动杆菌均携带bla_OXA-23,敏感株未检出携带该基因。RT-PCR法可检出转化bla_OXA-23/pYMAb3载体菌株表达bla_OXA-23基因,而转化pYMAb3空载体菌株未检出bla_OXA-23基因表达。转化bla_OXA-23/pYMAb3载体菌株对亚胺培南MIC为32μg/mL,转化空载体菌株MIC仅为0.5μg/mL。结论我院耐亚胺培南的鲍曼不动杆菌均携带bla_OXA-23基因,可以作为耐药菌株筛选的分子标记。由质粒携带的bla_OXA-23基因是诱发我院鲍曼不动杆菌耐药的重要原因,在院感工作中需高度重视。     

Resistance determinants in strains of Clostridium difficile from two geographically distinct populations

Ninety-three clinical isolates of Clostridium difficile, comprising 65 from Royal Gwent Hospital, Newport and 28 from Southmead Hospital, Bristol were examined to determine the prevalence of genes coding for macrolide resistance and to explore differences in susceptibility patterns. Antibiogram testing produced similar results for both sets of strains with respect to amoxicillin, tetracycline, erythromycin and cefotaxime. Results differed for rifampicin, where 53% of the Bristol isolates were resistant, compared with 3% of the Newport isolates. Clindamycin disc susceptibility testing produced similar resistance rates. However, clindamycin MIC determinations revealed that 53% of the Bristol strains exhibited high-level resistance (MIC > 256 mg/L), whereas strains from Newport had clindamycin MICs ranging from 0.25 to 3 mg/L. erm (B) was present in 15 of the strains from Bristol and in none of the Newport strains. erm (F) and erm (Q) were not detected in either population. The two geographically distinct populations of C. difficile differed considerably in their susceptibility to antibiotics. The possibility that C. difficile may serve as a conservator for resistant determinants subsequent to exposure to antimicrobial agents, has important implications for infection control.     

Antimicrobial activity of carbapenems and the combined effect with aminoglycoside against recent clinical isolates of Pseudomonas aeruginosa

The carbapenem susceptibility of 32 strains of Pseudomonas aeruginosa recently isolated in Kakogawa municipal hospital was investigated. The MIC ranges of imipenem, panipenem, and meropenem were 0.25-16 mg/L, 0.5-16 mg/L, and < 0.03-4 mg/L, respectively, and meropenem showed the highest antipseudomonal activity among the three carbapenems tested. In the analysis based on the MIC interpretive standards established by NCCLS, the resistance rates of test strains for imipenem, panipenem, and meropenem were 6.3%, 15.6%, and 0%, respectively. We also investigated the in vitro combined effect of imipenem or meropenem with amikacin against another 20 isolates of P. aeruginosa by checkerboard titration assay. Antagonism (minimum FIC index > 2) was not observed in any combinations against all strains tested. Super-additive effects (minimum FIC index < 1) in the combination of imipenem and amikacin were observed in eight (40%) strains tested. In contrast, in the combination of meropenem and amikacin, super-additive effects were observed in 14 isolates (70%). These results suggested that meropenem is superior to imipenem in combined effect with amikacin against P. aeruginosa. In conclusion, meropenem showed higher antipseudomonal activities than other carbapenems tested in both conditions, alone and in combination with amikacin. With regard to the clinical efficacy and prevention of antibiotic resistance, meropenem monotherapy or combination therapy with aminoglycoside is the most superior treatment for pseudomonal infections, and the findings in this study suggest that meropenem is still clinically very useful.