Heteroresistance to cephalosporins and penicillins in Acinetobacter baumannii

Heteroresistance to antimicrobial agents may affect susceptibility test results and therapeutic success. In this study, we investigated heteroresistance to cephalosporins and penicillins in Acinetobacter baumannii, a major pathogen causing nosocomial infections. Two A. baumannii isolates exhibited heteroresistance to ampicillin-sulbactam, ticarcillin-clavulanic acid, cefepime, and cefpirome, showing a distinct colony morphology of circular rings within the inhibition halos. Pulsed-field gel electrophoresis (PFGE) and outer membrane protein (OMP) analysis demonstrated that subpopulations around the disks/Etest strips and the original strains all belonged to the same PFGE type and OMP profile. Population analysis profile (PAP) showed the presence of heteroresistant subpopulations with high cefepime resistance levels in two isolates (008 and 328). Interestingly, A. baumannii 008 contained two peaks: one was grown in the presence of up to 1 μg of cefepime/ml, the other apparently occurred when the concentration of cefepime was raised to 256 μg/ml. After serial passages without exposure to cefepime, the PAP curve maintained the same trend observed for the original strain of A. baumannii 008. However, the PAP curve showed a shift to relatively lower cefepime resistance (from 256 to 64 μg/ml) in A. baumannii 328 after 10 passages in antibiotic-free Mueller-Hinton agar plates. Convergence to a monotypic resistance phenotype did not occur. Growth rate analysis revealed that slower growth in resistant subpopulations may provide a strategy against antibiotic challenge. To our knowledge, this is the first report of heteroresistance to cephalosporins and penicillins in A. baumannii.     

多耐药鲍氏不动杆菌对替加环素的耐药分析

目的探讨替加环素对多耐药鲍氏不动杆菌的体外抗菌活性。方法从2009年4月至2010年3月中山大学附属第三医院住院患者中分离41株多耐药鲍氏不动杆菌（multidrug-resistant Acineto bacterbaumannii,MDR-AB）,采用SIEMENS公司MicroScan Walk Away40全自动微生物鉴定仪鉴定菌株,用K-B法测定鲍氏不动杆菌对12种抗菌药物的敏感性。结果鲍氏不动杆菌主要分离自痰标本,占80.5%;病区主要分布在ICU（37%）和神经外科（22%）。除亚胺培南外,其余11种抗菌药物的非敏感率均大于80%。替加环素的非敏感率为80.4%,敏感率为19.5%;亚胺培南的敏感率最高,为36.6%,非敏感率为63.4%。结论多耐药鲍氏不动杆菌对替加环素的非敏感率很高,可能与外排泵系统有关。替加环素对MDR-ABA感染的治疗效果不容乐观。     

Modifications to CHROMagar Acinetobacter for improved selective growth of multi-drug resistant Acinetobacter baumannii

Performance of CHROMagar Acinetobacter was assessed for the selective isolation and identification of Acinetobacter baumannii. The medium was effective in suppressing the growth of other Gram-positive and Gram-negative species while the addition of KPC supplement ensured growth of only carbapenem resistant A baumannii.     

Two cases of necrotizing fasciitis due to Acinetobacter baumannii

Necrotizing fasciitis has conventionally been associated with the streptococci, and when it is caused by other organisms, it is most often the result of a polymicrobial infection. We report on two cases of fatal monomicrobial necrotizing fasciitis due to Acinetobacter baumannii, an unusual finding that may be an indication of enhanced virulence of the organism.     

Infective endocarditis due to Acinetobacter baumannii complex--a case report

We report a case of infective endocarditis caused by Acinetobacter baumannii complex in a 27-year-old male patient. The patient presented with fever of five days duration, palpitation, dyspnea, cough and chest pain. He had undergone a surgical repair of ruptured aneurysm of sinus of valsalva a month before. The transthoracic echocardiogram revealed a large vegetation on the aortic valve. Three samples of blood for culture grew gram-negative pleomorphic coccobacilli within 24 hours which were identified by cultural and biochemical characteristics to be Acinetobacter baumannii complex. Antimicrobial susceptibility was performed by Kirby-Bauer method and the isolate were found to be resistant to ampicillin, Ciprofloxacin, Ceftriaxone, Gentamicin, Amikacin, Augmentin, Levofloxacin, Piperacillin-Tazobactam, Netilimicin and sensitive to Imipenem. Patient was initially treated with Ceftraixone and Gentamicin and subsequently with Ampicillin and Amikacin but did not respond to treatment and died of sepsis before therapy with Imipenem could be started.     

Acinetobacter baumannii colonization in ventilated preterm infants

The role ofAcinetobacter baumannii in infections in ventilated preterm infants was evaluated in 15 colonized infants (11 male, 4 female) in a pediatric intensive care unit. These cases were randomly matched by birth weight and gestational age with ventilated non-colonized controls (8 male, 7 female). Case records were reviewed for signs and symptoms of infection. Colonized infants were ventilated significantly longer (p<0.05) than controls, and had body temperatures of >37°C for a significantly longer period of time (p<0.05). No other parameter of infection differed significantly between the groups. The duration of intensive care treatment did not differ between cases and controls, nor did the weight gain during intensive care treatment. No fatalities occurred in either group.     

OXA beta-lactamase-mediated carbapenem resistance in Acinetobacter baumannii

Objectives: Acinetobacter baumannii is a significant pathogen in health care settings. In recent years, an increase in carbapenem resistance among A. baumannii due to Ambler class B metallo-beta-lactamases or class D OXA carbapenamases has been reported. In this study we detected the presence of OXA carbapenamases and coproduction of metallo-beta-lactamases (bla_VIMand bla_IMP) by phenotypic and genotypic methods in carbapenem resistant clinical isolates of Acinetobacter baumannii. Materials and Methods: A total of 116 consecutive, non-duplicate carbapenem resistant A. baumannii isolated from various clinical specimens were included in the study. The modified Hodge test and inhibitor potentiated disk diffusion tests were done for the screening of carbapenamase and metallo-beta-lactamase production, respectively. Polymerase chain reaction (PCR) was performed for the detection of OXA (bla_{OXA 23} like, bla_OXA-24 like, bla_OXA-51 like and bla_OXA-58 like genes) and metallo-beta-lactamases (bla_VIMand bla_IMP) genes. Gene sequencing was performed for representative isolates. Results: Among 116 A. baumannii, OXA genes were detected in 106 isolates. Bla_{OXA 51} like (n = 99) and bla_{OXA -23} like (n = 95) were the most common and they coexisted in 89 isolates. bla_OXA-24 like gene was detected in two isolates of which one also carried bla_OXA-51 like and bla_OXA-58 like genes. The modified Hodge test was positive in 113 isolates. The metallo-beta-lactamase screening test was positive in 92 isolates. bla_vimwas detected in 54 isolates of which 1 also carried the bla_IMP gene. Conclusions: bla_OXA-23 like and bla _{OXA 51} like genes are the most common types of OXA carbapenamases while the bla_VIMtype is the most common type of metallo-beta-lactamase contributing to carbapenem resistance in clinical isolates of A. baumannii. The coproduction of OXA and metallo-beta-lactamases is not an uncommon phenomenon in A. baumannii.     

鲍曼不动杆菌对碳青霉烯类抗生素的耐药分析及基因型研究

目的分析20株鲍曼不动杆菌对耐碳青霉烯类抗生素的耐药性及对碳青霉烯酶基因的研究。方法用API鉴定条进行细菌鉴定及K-B法进行药敏试验,用碳青霉烯酶4种基因的特异性引物进行聚合酶链反应（PCR）扩增和基因型的测序分析,并通过网上Genbank进行比对以确定编码酶基因的类型。结果 20株鲍曼不动杆菌对左旋氧氟沙星、丁胺卡那霉素、多粘菌素B的耐药率分别为50%、25%、4%。其它抗生素的耐药率均在90%以上。携带D类碳青霉烯酶OXA-23基因有17株（85%）,携带OXA-51基因有15株（75%）,OXA-24、OXA-58基因引物PCR扩增为阴性,随机各抽取3株OXA-23基因阳性株进行测序后通过在网上Genbank比对发现与OXA-23标准株99%同源,OXA-51基因阳性株与OXA-51标准株98%同源。结论本院耐碳青霉烯类抗生素的鲍曼不动杆菌对多粘菌素B的耐药率最低,其次是丁胺卡那霉素,以携带OXA-23型碳青霉烯酶基因为主,应引起临床高度重视,防止在院内广泛传播。     

Adherence of Acinetobacter baumannii strains to human bronchial epithelial cells

Acinetobacter baumannii is an important nosocomial pathogen, but the mechanisms contributing to its epidemicity and virulence are largely unknown. The organism is able to colonize skin and mucosal surfaces of the human host. Adherence of microorganisms to host cells is an important virulence factor as it is the initial step of the colonization process. In the present study, adherence of A. baumannii to human bronchial epithelial NCI-H(292) cells was examined by light and scanning electron microscopy. Thirty-seven strains were investigated including 18 from outbreaks, 16 not associated with outbreaks, and three for which an epidemic implication was unknown. Eight and 11 isolates belonged to European clone I and II, respectively. Two types of adherence were observed, dispersed adherence of bacteria to the cell, and adherence of clusters of bacteria at localized areas of the cells. Bacteria with dispersed adherence interacted with the epithelial cells through fimbriae, but were also entrapped by protrusions extending from the epithelial cells. Quantitative adherence varied considerably among strains but there was no significant correlation of the outbreak-associated strains with the percentage of infected cells. There was, however, a correlation between the clonal lineage and the percent of infected cells, with clone II being more adherent than clone I (P<0.05). Ten consecutive isolates from one outbreak were investigated to test whether adherence increased during passage among patients, but this appeared not to be the case. This study showed that A. baumannii adheres to human bronchial epithelial cells in vitro and that A. baumannii strains of clone II had a relatively high capacity for adhering to these cells.     

鲍曼不动杆菌临床株的外排泵研究

目的 探讨鲍曼不动杆菌临床株外排泵AdeABC、AdeIJK、AdeFGH、AbeM、AbeS、CraA、MdtL的表达与耐药的关系.方法 收集多重耐药鲍曼不动杆菌临床株32株和敏感株10株,PCR扩增泵基因;选取主要克隆型的21株多重耐药株和10株敏感株,实时荧光定量RT-PCR方法检测泵基因adeB、adeJ、adeG、abeM、abeS、craA、mdtL的mRNA相对表达水平,PCR扩增泵调控基因adeRS、adeL并测序分析.结果 32株多重耐药鲍曼不动杆菌临床株中携带泵结构基因片段adeB100％、adeJ 100％、adeG 100％、abeM 96.88％、abeS 100％、craA 100％、mdtL 93.75％,10株敏感株均存在7种泵结构基因片段;主要克隆型的21株多重耐药株和10株敏感株adeB、abeM、mdtL的mRNA相对表达水平的差异有统计学意义( P＜0.001,P=0.001,P=0.013),选取多重耐药株AbR3和AbR11检测外排泵AdeABC调控基因adeRS序列出现氨基酸替代及缺失,而外排泵AdeFGH调控基因adeL序列无基因突变.结论 鲍曼不动杆菌临床株外排泵AdeABC、AbeM、MdtL的表达可能与耐药性有关.     

A PCR-based method to differentiate between Acinetobacter baumannii and Acinetobacter genomic species 13TU

A new PCR-based method that exploits differences in gyrB gene sequences was developed to distinguish between Acinetobacter baumannii and Acinetobacter genomic sp. 13TU. Among 118 clinical and reference Acinetobacter strains, 102 of which were previously speciated by amplified rDNA restriction analysis as belonging to the Acinetobacter calcoaceticus-A. baumannii complex, the method correctly identified 31 A. baumannii and 54 Acinetobacter genomic sp. 13TU isolates to the species level. The method was rapid, specific and easy to interpret.     

Detection of carbapenemase-producing Acinetobacter baumannii in a hospital

Acinetobacter baumannii strains resistant to both imipenem (IPM) and ceftazidime (CAZ) were isolated from 1994 through 1996 at Gunma University Hospital. Nine isolates from different inpatients were examined for carbapenem-hydrolyzing activity and for the carbapemase gene bla(IMP) by the PCR method. All nine isolates were carbapenemase-producing strains that hydrolyzed IPM and that harbored bla(IMP). The bla(IMP) gene was transmissible by conjugation to an IPM-susceptible recipient strain of A. baumannii and conferred resistance to IPM, CAZ, cefotaxime (CTX), ampicillin (AMP), and piperacillin (PIP). Either intermediate or high-level resistance to amikacin (AMK) was transferred from two and five strains, respectively, concomitantly with bla(IMP), and gentamicin (GEN) resistance was also transferred in one instance of high-level AMK resistance. Comparative examination of clinical isolates for resistance patterns to nine drugs, IPM, CAZ, CTX, aztreonam, AMP, PIP, AMK, GEN, and norfloxacin, in addition to pulsed-field gel electrophoresis patterns with NotI-digested genomic DNA, confirmed nosocomial transmission of infections involving carbapenemase-producing A. baumannii strains.     

Predictors of mortality in Acinetobacter baumannii bacteremia.

This study retrospectively investigated 149 episodes of Acinetobacter baumannii bacteremia which occurred during a 41-month period from September 1997 to January 2001. Bacteremia was nosocomial in 139 (93%) of the episodes and community-acquired in 10 (7%). Thirty three deaths (22.1%) were attributed to these episodes of A. baumannii bacteremia. The mean age of survivors was younger than that of patients who died of bacteremia (60.4 +/- 19.9 vs 67.1 +/- 17.4) but this result was not significant on univariate analysis (p=0.084). Previous intensive care unit stay was longer among survivors than among patients who died of bacteremia (9.5 vs 18 days, p=0.048). Factors associated with mortality included immunosuppression (p=0.019), shock (p=0.002), recent surgery (p=0.008), invasive procedures such as central venous catheterization (p=0.002), urinary catheterization (p=0.012), placement of a nasogastric tube (p<0.001), pulmonary catheterization (p=0.015), and mechanical ventilation (p=0.035). The number of underlying conditions (p=0.015) and invasive procedures (p<0.001) were positively correlated with mortality. Mortality was significantly associated with lower platelet count (p=0.001) and lower serum albumin concentration (p=0.005). Patients with catheter-related bacteremia had a high survival rate (96.2%), while survival rate was low in patients with infection originating from the respiratory tract (60.8%). Susceptibility testing by agar dilution test indicated that imipenem was the most effective antibiotic, followed by cefepime and ciprofloxacin. The mortality rate was lower in patients who received 1 or more antibiotics to which the isolates were susceptible, but this difference was not significant (p=0.197). On multivariate analysis, factors that independently correlated with mortality were increased age (p=0.003), immunosuppressive status (p=0.001), recent surgery (p=0.002), acute respiratory failure (p=0.004), acute renal failure (p=0.009) and septic shock (p<0.001). These findings highlight the importance of a treatment strategy based on risk stratification among patients with A. baumannii bacteremia.     

Heteroresistance to vancomycin in Enterococcus faecium

This study presents the first report of vancomycin heteroresistance in an Enterococcus faecium isolate from a patient. The original isolate was susceptible in vitro to vancomycin. E-tests showed growth of subcolonies in a zone of inhibition with a vancomycin MIC of >256 microg/ml. Both the susceptible and resistant colonies were from the same strain as determined by PFGE, and both contained the vanA gene as determined by PCR.     

Plasmid DNA fingerprinting of Acinetobacter species other than Acinetobacter baumannii.

During the last years Acinetobacter species have emerged as clinically significant pathogens. Most infections are nosocomially acquired and mainly due to Acinetobacter baumannii. Little is known about the epidemiology and clinical significance of unnamed Acinetobacter species 3 (the second most often encountered member of the genus Acinetobacter) and other Acinetobacter species such as A. johnsonii, A. junii, and A. lwoffii. Seventy-five clinical isolates of Acinetobacter species other than A. baumannii (Acinetobacter species 3, n = 37; A. johnsonii, n = 20; A. junii, n = 8; A. lwoffii, n = 10) recovered from 66 patients over a period of 12 months were analyzed by plasmid DNA fingerprinting. Plasmids were found in 84.4% of Acinetobacter species 3 isolates and in all A. johnsonii, A. junii, and A. lwoffii isolates. Strains harbored up to 15 plasmids each. Almost every isolate gave a unique plasmid pattern. With one exception, identical plasmid profiles were detected only in corresponding isolates recovered from blood cultures and intravascular catheters from a given patient. Plasmid DNA fingerprinting proved to be useful for typing Acinetobacter species other than A. baumannii. There was no evidence of patient-to-patient transmission or hospital outbreaks due to these species. This finding is in contrast to the results obtained in studies of the hospital epidemiology of A. baumannii.     

Characteristics of Meropenem Heteroresistance in Klebsiella pneumoniae Carbapenemase (KPC)-Producing Clinical Isolates of K. pneumoniae

Meropenem heteroresistance was investigated in six apparently meropenem-susceptible, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) clinical isolates, compared with that in carbapenemase-negative, meropenem-susceptible controls. In population analyses, the KPC-KP isolates grew at meropenem concentrations of 64 to 256 μg/ml. Heteroresistant colonies had significantly elevated expression of the bla_KPC gene compared with the native populations but did not retain heteroresistance when subcultured in drug-free media. Time-kill assays indicated that meropenem alone was not bactericidal against KPC-KP but efficiently killed the control strains.Since the beginning of the last decade, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) isolates have been increasingly detected in the United States and subsequently in several regions worldwide (3, 4, 13, 17, 21). KPC enzymes efficiently hydrolyze all β-lactam molecules (1, 22), conferring various levels of resistance to all β-lactam compounds, including carbapenems (13). However, KPC-producing K. pneumoniae may appear susceptible to carbapenems, mainly meropenem (2, 13), by reference CLSI agar dilution or broth microdilution methods as well as by automated systems (6, 15, 17). Characteristically, it has been reported that automated systems may identify as many as 87% of KPC-KP isolates to be susceptible to meropenem (13). The detection of the susceptibility level of KPC-KP isolates to carbapenems has been shown to be difficult due to the phenotypic heterogeneity that they commonly exhibit (3, 10, 13). For instance, in agar diffusion methods such as disk diffusion or Etest, the heterogeneous growth to carbapenems of KPC-KP results in the appearance of scattered colonies within the inhibition zones (9, 13).These issues raise the need for cautious evaluation of susceptibility testing in KPC-KP isolates that are recovered in clinical laboratories. In our clinical laboratories, several KPC-KP isolates that appear susceptible by automated susceptibility assays or reference dilution assays contain heterogeneous subpopulations (D. Sofianou and K. Themeli-Digalaki, personal communications). It has been also shown that among Greek KPC-KP isolates, meropenem tends to exhibit lower MICs than imipenem or ertapenem (17, 20). In that respect, the aim of the present study was to characterize the heterogeneous mode of growth of apparently meropenem-susceptible KPC-KP clinical isolates by population analyses and bactericidal assays.