Effects of inoculum, delayed loading, and storage temperature of blood culture bottles on the detection of Burkholderia pseudomallei

Detection of Burkholderia pseudomallei in blood culture is critical to prompt appropriate treatment. Various factors may affect culture positivity. This study evaluated effects of various inocula (0.2-100 CFU/mL), delayed bottle loading periods (12-48 h), and storage temperatures (ambient, 4 and 35 degrees C) for the detection of B. pseudomallei by the BacT/ALERT system. The results suggested that bottles may be preincubated at 35 degrees C for up to 12 h, or held at ambient temperature for up to 24 h, or refrigerated at 4 degrees C for up to 48 h.     

Liver abscess caused by Burkholderia pseudomallei in a young man: A case report and review of literature

Pyogenic liver abscess is a common entity in Indian subcontinent and is mostly caused by gram negative bacteria. Melioidosis is not commonly seen in India and only a few cases are reported. It can give rise to multiple abscesses at different sites including liver. We report a case of isolated liver abscess caused by Burkholderia pseudomallei (B. pseudomallei) in a 29-year-old recently diagnosed diabetic, immunocompetent male. Diagnosis was made by imaging and culture of pus aspirated from the abscess and he was treated with percutaneous pigtail catheter drainage followed by antibiotics (meropenem and trimethoprim-sulphmethoxazole). Melioidosis is an emerging infection in India and has high mortality rate, so early diagnosis and prompt management is warranted which requires clinical vigilance and an intensive microbiological workup. Clinicians should be aware of isolated liver abscess caused by B. pseudomallei in appropriate clinical settings.     

Improved diagnosis of melioidosis using a 2-dimensional immunoarray

Melioidosis is caused by the Gram negative bacterium Burkholderia pseudomallei. The gold standard for diagnosis is culture, which requires at least 3–4 days obtaining a result, hindering successful treatment of acute disease. The existing indirect haemagglutination assay (IHA) has several disadvantages, in that approximately half of patients later confirmed culture positive are not diagnosed at presentation and a subset of patients are persistently seronegative. We have developed 2 serological assays, an enzyme-linked immunosorbent assay (ELISA), and a 2-dimensional immunoarray (2DIA), capable of detecting antibodies in patient sera from a greater proportion of IHA-negative patient subsets. The 2DIA format can distinguish between different LPS serotypes. Currently, the 2DIA has a sensitivity and specificity of 100% and 87.1%, respectively, with 100% of culture-positive, IHA-negative samples detected. The ELISA has a sensitivity and specificity of 86.2% and 93.5%, respectively, detecting 67% of culture-positive, IHA-negative samples. The ELISA and 2DIA tests described here are more rapid and reliable for serological testing compared to the existing IHA.     

Accuracy of commercial systems for identification of Burkholderia pseudomallei versus Burkholderia cepacia

Infection caused by Burkholderia pseudomallei or Burkholderia cepacia may result in fatal outcome unless the causative agent is accurately identified in a short period, which is critical for treatment. We evaluated the reliability of the commonly used commercial systems, API 20NE, VITEK 2, and WalkAway 96, for comparative identification of B. pseudomallei versus B. cepacia clinical isolates. Based on biochemical and molecular tests as reference methods, API 20NE was probably the most reliable, with an accuracy of 87% and 93%, for the identification of B. pseudomallei and B. cepacia, respectively. The VITEK 2 and WalkAway 96 systems resulted in a number of misidentification and, thus, were less reliable. The performance of each system and identification guidelines for B. pseudomallei and B. cepacia are discussed. Our study emphasized that laboratories should carefully interpret the identification of B. pseudomallei and B. cepacia when using commercial systems.     

A Melioidosis Patient Presenting with Brainstem Signs in the Emergency Department

Background

Neurological abnormalities in melioidosis are rare but may manifest as an acute stroke, and in the emergency department (ED), an inappropriate stroke treatment may threaten a patient’s life.

Objectives

A case of cerebral melioidosis is reported in a patient presenting with brainstem signs to increase awareness of the uncommon presentations of melioidosis that may cause a delayed diagnosis in the ED.

Case Report

A 45-year-old man who worked as a construction worker, with diabetes mellitus and alcoholic liver cirrhosis, presented to the ED after a 10-day period of fever and cough. He was initially diagnosed and treated as a case of community-acquired pneumonia. However, a sudden change in consciousness with 6th and 7th cranial nerve palsy and flaccid paralysis were noted while he was in the ED, and acute brainstem stroke was suspected. Brain magnetic resonance imaging disclosed brainstem lesions, slightly hypointense on T1-weighted images and hyperintense on T2-weighted images. Blood and urine cultures subsequently yielded Burkholderia pseudomallei. Abdominal computed tomography revealed multiple small consolidated patches, ground-glass opacities, small nodules in the lower lungs bilaterally, and a pancreatic tail abscess. Systemic melioidosis with lung, pancreas, urogenic tract, and brainstem involvement was diagnosed. Three weeks after admission, the patient died from a sudden onset of apnea and asystole.

Conclusions

In light of this case, patients with identifiable risk factors, especially underlying diabetes, a history of positive soil contact, and those who lived in an endemic area or ever traveled to an endemic area, and who present themselves with fever and neurologic deficit or multi-organ involvement, should have melioidosis considered in the differential diagnosis.     

Clinical characteristics and outcomes of patients with Burkholderia cepacia bacteremia in an intensive care unit

The purpose of this study was to investigate a cohort of patients with Burkholderia cepacia bacteremia in the intensive care unit (ICU) at our institution. A large outbreak of B. cepacia bacteremia involving 95 patients lasted for 4 years in an ICU in northern Taiwan. The clinical characteristics and antimicrobial treatment responses of these patients were analyzed. Minimal inhibitory concentrations were determined and pulse-field gel electrophoresis was performed for the 73 available isolates. Overall, the in-hospital mortality rate was 53.8% and the 14-day mortality rate was 16.8%. Most patients (95.6%) had several underlying diseases and all but 1 patient had tracheal intubation. Malignancy (37.5% versus 13.9%, P = 0.02) and higher Sequential Organ Failure Assessment (SOFA) scores at the onset of bacteremia (11.9 ± 4.7 versus 7.9 ± 3.6, P < 0.001) were significant risk factors for 14-day mortality. In contrast, treatment with ceftazidime (76.0% versus 43.7%, P = 0.02) and diabetes (51.9% versus 13.8%, P = 0.01) were associated with decreased mortality. In the multivariate analysis, malignancy and higher SOFA score were significant risk factors for mortality [odds ratio (OR) 12.45, 95% confidence interval (CI) 2.35-65.94; OR 1.20, 95% CI 1.00-1.45, respectively]. Meropenem, ceftazidime, and piperacillin-tazobactam were the most active agents (susceptible rate 100%, 97.3%, and 97.3%, respectively). Pulsed-field gel electrophoresis results indicated 49 of the 73 isolates could be classified as outbreak-related strains. There was no significant difference in the clinical characteristics and outcomes of patients with bacteremia due to outbreak-related and non-outbreak-related strains. In conclusion, malignancy and a higher SOFA score at onset of bacteremia predicted increased mortality, but the clinical presentation and outcome of patients with outbreak and non-outbreak strains were similar.     

ELISA and immuno–polymerase chain reaction assays for the sensitive detection of melioidosis

Melioidosis is caused by the Gram-negative bacterium Burkholderia pseudomallei. The gold standard for diagnosis is culture, which requires at least 3–4 days to obtain a result, hindering successful treatment of acute disease. An indirect haemagglutination assay (IHA) is often used but lacks sensitivity. Approximately half of patients later confirmed culture positive are not detected by IHA at presentation and a subset of patients persistently continue to be IHA negative. More rapid and reliable serologic testing for melioidosis is essential and will improve diagnosis and patient outcome. We have developed an ELISA and a quantitative immuno-polymerase chain reaction assay capable of detecting melioidosis-specific antibodies and demonstrate their validity with IHA-negative sera from patients with melioidosis. These new sensitive assays are based upon a secreted antigenic fraction from B. pseudomallei and will be ideal for the diagnosis of melioidosis in patients in nonendemic regions returning from endemic tropical areas and for seroepidemiologic surveys.     

强台风后类鼻疽发病情况及临床菌株分子特征的初步分析

目的 了解强台风袭击海南省后灾区类鼻疽病的发病情况及其临床菌株的分子特征。方法 2014年7月威马逊台风袭击海南省后,于3所大型医疗机构开展类鼻疽新发病例主动监测工作。对收集的病例绘制分布地图,并对发病情况及临床表现等进行描述性流行病学分析,采用多位点序列分型（MLST）、脉冲场凝胶电泳（PFGE）、多位点可变数目串联重复序列多态性分析（MLVA）等方法对病例临床分离株进行分子溯源及同源性比较。结果 调查期间共发现类鼻疽确诊病例16例,高于2013年同期病例数（7人）,其中12例（75.0%）于灾后4周内发病,其余4例于5～8周出现。病例均来自受灾严重的海南省北部地区,其中9例集中分布于台风中心途经的文昌-海口带状区域。临床表现以类鼻疽肺炎（75.0%）、类鼻疽败血症（68.8%）为主;病死率达50.0%。分子分型显示16株类鼻疽伯克霍尔德菌区分为12个序列型（ST）、13种PFGE带型及15个MLVA型别,除两株ST1325型菌株外来源均不相同。结论 威马逊台风过后2个月内海南省灾区出现聚集性类鼻疽病例;相关类鼻疽伯克霍尔德菌对应多个ST型,呈高度多态性,能够排除生物恐怖袭击的可能性。出现聚集性类鼻疽病例的原因及其与台风的关系尚需进一步调查。     

Detection of the host immune response to Burkholderia mallei heat-shock proteins GroEL and DnaK in a glanders patient and infected mice

We examined, by enzyme-linked immunosorbent assay and Western blot analysis, the host immune response to 2 heat-shock proteins (hsps) in a patient and mice previously infected with Burkholderia mallei. The patient was the first reported human glanders case in 50 years in the United States. The expression of the groEL and dnaK operons appeared to be dependent upon a sigma(32) RNA polymerase as suggested by conserved heat-shock promoter sequences, and the groESL operon may be negatively regulated by a controlling invert repeat of chaperone expression (CIRCE) site. In the antisera, the GroEL protein was found to be more immunoreactive than the DnaK protein in both a human patient and mice previously infected with B. mallei. Examination of the supernatant of a growing culture of B. mallei showed that more GroEL protein than DnaK protein was released from the cell. This may occur similarly within an infected host causing an elevated host immune response to the B. mallei hsps.     

Prevalence of quinolone resistance determinants in non-typhoidal Salmonella isolates from human origin in Extremadura,Spain

Resistance to the quinolones nalidixic acid (NAL) and ciprofloxacin (CIP) and the occurrence of quinolone resistance determinants have been investigated in 300 non-typhoidal Salmonella from human origin, isolated in the years between 2004 and 2008, in 6 hospitals within Extremadura (Spain). Salmonella Enteritidis was the major serotype found among quinolone-resistant isolates, most of which were clustered by clonal analysis to a single clone, which presented D87 or S83 substitutions in GyrA. Eleven isolates presented the non-classical quinolone resistance phenotype (resistance to CIP and susceptibility to NAL), lacking mutations in the quinolone resistance determinant region of topoisomerase genes. Among them, one Salmonella Typhimurium isolate carried a qnrS1 gene in a low-molecular-weight plasmid, pQnrS1-HLR25, identical to plasmids previously found in the UK, Taiwan, and USA. The occurrence of this genetic element could represent a risk for the horizontal transmission of quinolone resistance among Enterobacteriaceae in the Iberian Peninsula.     

Research on Candida dubliniensis in a Brazilian yeast collection obtained from cardiac transplant, tuberculosis, and HIV-positive patients, and evaluation of phenotypic tests using agar screening methods

The aim of this study was to research Candida dubliniensis among isolates present in a Brazilian yeast collection and to evaluate the main phenotypic methods for discrimination between C. albicans and C. dubliniensis from oral cavity. A total of 200 isolates, presumptively identified as C. albicans or C. dubliniensis obtained from heart transplant patients under immunosuppressive therapy, tuberculosis patients under antibiotic therapy, HIV-positive patients under antiretroviral therapy, and healthy subjects, were analyzed using the following phenotypic tests: formation and structural arrangement of chlamydospores on corn meal agar, casein agar, tobacco agar, and sunflower seed agar; growth at 45 °C; and germ tube formation. All strains were analyzed by polymerase chain reaction (PCR). In a preliminary screen for C. dubliniensis, 48 of the 200 isolates on corn meal agar, 30 of the 200 on casein agar, 16 of the 200 on tobacco agar, and 15 of the 200 on sunflower seed agar produced chlamydoconidia; 27 of the 200 isolates showed no or poor growth at 45 °C. All isolates were positive for germ tube formation. These isolates were considered suggestive of C. dubliniensis. All of them were subjected to PCR analysis using C. dubliniensis-specific primers. C. dubliniensis isolates were not found. C. dubliniensis isolates were not recovered in this study done with immunocompromised patients. Sunflower seed agar was the medium with the smallest number of isolates of C. albicans suggestive of C. dubliniensis. None of the phenotypic methods was 100% effective for discrimination between C. albicans and C. dubliniensis.     

Macrolide and tetracycline resistance among Moraxella catarrhalis isolates from 2009 to 2011

The activity of macrolides and other antimicrobials was evaluated by Clinical and Laboratory Standards Institute reference broth microdilution methods for Moraxella catarrhalis isolates collected during 2009-2011. In Europe, the United States, and Latin America, M. catarrhalis resistance to macrolides and tetracycline was <1%. However, in the Asia-Pacific region, clarithromycin and tetracycline resistance was 7.6% and 3.2%, respectively. The higher resistance rate in the Asia-Pacific region to clarithromycin and tetracycline was primarily due to isolates from multiple locations in China.     

Strains of Burkholderia cenocepacia genomovar IIIA possessing the cblA gene that are distinct from ET12

Three strains of Burkholderia cenocepacia genomovar IIIA that were polymerase chain reaction positive for cblA, bcrA, and the epidemic strain marker, but were distinct from representatives of ET12 by pulsed-field gel electrophoresis, are described. One of these strains was shown to express cable pili by electron microscopy.     

Prevalence and mechanisms of decreased susceptibility to carbapenems in Klebsiella pneumoniae isolates

In this study, we examined the prevalence of and mechanisms of decreased susceptibility to either imipenem or meropenem in Klebsiella pneumoniae isolates. A total of 230 clinical isolates of K. pneumoniae were collected from 13 clinical laboratories from a nationwide distribution. The MICs of imipenem and meropenem were determined by the agar dilution method. To characterize the isolates with decreased susceptibility to carbapenems (MICs of >2 microg/mL), we performed polymerase chain reaction amplification of a variety of beta-lactamase genes, isoelectric focusing, and outer membrane profile analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. Three isolates (BD6, BD8, and KN16) exhibited decreased susceptibility to carbapenems with imipenem MICs of 1, 4, and 8 microg/mL and meropenem MICs of 4, 8, and 4, respectively. Isolate BD6 produced bla(TEM-1), bla(SHV-12), and bla(OXA-17); isolate BD8 produced bla(GES-3), bla(SHV-12), and bla(OXA-17); and isolate KN16 produced bla(TEM-11), bla(SHV-12), and bla(DHA-1). In all the 3 isolates, OmpK35 porin was not expressed, and in 1 isolate (KN16), OmpK36 was not expressed either. The prevalence of decreased susceptibility to carbapenems was low (1.3%), and none of them showed overt resistance to carbapenems. Decreased susceptibility to carbapenems can occur in K. pneumoniae when bla(GES-3), bla(TEM-11), bla(SHV-12), bla(OXA-17), and/or bla(DHA-1) are produced in combination with porin loss. In addition, to our knowledge, this is the 1st report of bla(OXA-17) in Enterobacteriaceae.     

Acinetobacter species isolates from a range of environments: species survey and observations of antimicrobial resistance

Acinetobacter species isolates from a range of environments, including soil, were investigated. We determined 16S rRNA and rpoB gene sequences for species identification and performed tests of antimicrobial resistance susceptibility. Twenty-nine of the isolates (8 from soil and 21 from life environment) belonged to the genus Acinetobacter. Fourteen Acinetobacter species were identified among 29 isolates: 4 A. baumannii, 3 A. calcoaceticus, 1 A. nosocomialis, 2 A. pittii, and 2 Acinetobacter gen. sp. 'close to 13TU' as A. calcoaceticus-baumannii complex. Three Acinetobacter species isolates were identified as novel species candidates. Three Acinetobacter species isolates were resistant to imipenem: 1 A. parvus and 2 novel species candidates of Acinetobacter. Eight isolates showed resistance to colistin: all Acinetobacter gen. sp. 'close to 13TU' (2 isolates) and A. parvus isolates (3 isolates) were resistant to colistin. Although the genotypes of A. baumannii isolates from various natural environments were different from those of clinical isolates, the presence of clinically important and antimicrobial resistant Acinetobacter species in the natural environment may represent a threat to public health.     

Molecular characterization of fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates from Shanghai, China

China is one of the countries with the highest prevalence of fluoroquinolone-resistant (FQ(r)) Mycobacterium tuberculosis. Nevertheless, knowledge on the molecular characterization of the FQ(r)M. tuberculosis strains of this region remains very limited. This study was performed to investigate the frequencies and types of mutations present in FQ(r)M. tuberculosis clinical isolates collected in Shanghai, China. A total of 206 FQ(r)M. tuberculosis strains and 21 ofloxacin-sensitive (FQ(s)) M. tuberculosis strains were isolated from patients with pulmonary tuberculosis in Shanghai. The phenotypic drug susceptibilities were determined by the proportion method, and the mutations inside quinolone resistance-determining region (QRDR) of gyrA and gyrB genes were identified by DNA sequence analyses. Among 206 FQ(r)M. tuberculosis strains, 44% (90/206) were multidrug-resistant isolates and 39% (81/206) were extensively drug-resistant isolates. Only 9% (19/206) were monoresistant to ofloxacin. In total, 79.1% (163/206) of FQ(r) isolates harboured mutations in either gyrA or gyrB QRDR. Mutations in gyrA QRDR were found in 75.7% (156/206) of FQ(r) clinical isolates. Among those gyrA mutants, a majority (75.6%) harboured mutations at amino acid position 94, with D94G being the most frequent amino acid substitution. Mutations in gyrA QRDR showed 100% positive predictive value for FQ(r)M. tuberculosis in China. Mutations in gyrB were observed in 15.5% (32/206) of FQ(r) clinical isolates. Ten novel mutations were identified in gyrB. However, most of them also harboured mutations in gyrA, limiting their contribution to FQ(r) resistance in M. tuberculosis. Our findings indicated that, similar to other geographic regions, mutations in gyrA were shown to be the major mechanism of FQ(r) resistance in M. tuberculosis isolates. The mutations in gyrA QRDR can be a good molecular surrogate marker for detecting FQ(r)M. tuberculosis in China.     

Molecular characterization of Italian Candida parapsilosis isolates reveals the cryptic presence of the newly described species Candida orthopsilosis in blood cultures from newborns

The authors report the molecular characterization of Candida parapsilosis isolates recovered from the blood and venous central catheter tips of patients admitted to different care units of the Polyclinic Hospital, University of Messina, Italy. Among 97 presumed C. parapsilosis isolates examined, 94 were identified as C. parapsilosis sensu stricto and the remaining 3 isolates were found to belong to the cryptic species Candida orthopsilosis which was recovered only from blood cultures of neonates (<30 days old) born prematurely. No C. metapsilosis was found in this study. This study emphasizes the role of C. parapsilosis as an important nosocomial pathogen, and it also describes, for the first time, the occurrence of C. orthopsilosis in newborns.     

Antimicrobial activity of ceftaroline and comparator agents tested against bacterial isolates causing skin and soft tissue infections and community-acquired respiratory tract infections isolated from the Asia-Pacific region and South Africa (2010)

Ceftaroline, the active metabolite of the prodrug ceftaroline fosamil, is a cephalosporin with in vitro bactericidal activity against resistant Gram-positive organisms including methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant strains of Streptococcus pneumoniae, and common Gram-negative organisms, including wild-type Enterobacteriaceae. We evaluated the in vitro activity of ceftaroline and selected comparator agents against bacterial isolates collected from patients with skin and soft tissue infections (SSTI) and community-acquired respiratory tract infections (CARTI) in the Asia-Pacific region and South Africa. A total of 2351 isolates, 1100 from SSTI and 1251 from CARTI, were collected from 25 medical centers distributed across 8 countries as part of the 2010 AWARE ceftaroline surveillance program and tested for susceptibility by reference broth microdilution methods. Ceftaroline was very active against S. aureus (MIC_50/90, 0.25/1 μg/mL; 93.4% susceptible), including MRSA (MIC_50/90, 1/2 μg/mL; 80.6% susceptible). Against β-hemolytic streptococci, ceftaroline demonstrated greater activity (MIC₉₀, 0.015 μg/mL) than penicillin (MIC₉₀, 0.06 μg/mL). Ceftaroline was also highly active against viridans group streptococci (MIC₉₀, 0.12 μg/mL). Similarly to ceftriaxone, ceftaroline activity against Escherichia coli (MIC_50/90, >32/>32 μg/mL) and Klebsiella spp. (MIC_50/90, 0.12/>32 μg/mL) was compromised by the high prevalence of isolates with an ESBL phenotype in the region, particularly in China. Ceftaroline was the most potent β-lactam tested against S. pneumoniae (MIC_50/90 of 0.015/0.25 μg/mL; 99.8% susceptible by Clinical and Laboratory Standards Institute [CLSI] criteria), and it was also highly potent against Haemophilus influenzae (MIC_50/90, ≤0.008/0.03 μg/mL; 100% susceptible by CLSI criteria). Ceftaroline was also active against H. parainfluenzae (MIC_50/90, ≤0.008/0.015 μg/mL) and Moraxella catarrhalis (MIC_50/90, 0.06/0.12 μg/mL). In summary, ceftaroline showed potent in vitro activity against a large collection of bacterial isolates (2351) associated with SSTI and CARTI from the Asia-Pacific region and South Africa.