Characterization of biofilm production in different strains of Acinetobacter baumannii and the effects of chemical compounds on biofilm formation.

Acinetobacter baumannii, an important emerging pathogen of nosocomial infections, is known for its ability to form biofilms. Biofilm formation increases the survival rate of A. baumannii on dry surfaces and may contribute to its persistence in the hospital environment, which increases the probability of nosocomial infections and outbreaks. This study was undertaken to characterize the biofilm production of different strains of A. baumannii and the effects of chemical compounds, especially antibiotics, on biofilm formation. In this study, no statistically significant relationship was observed between the ability to form a biofilm and the antimicrobial susceptibility of the A. baumannii clinical isolates. Biofilm formation caused by A. baumannii ATCC 17978 after gene knockout of two-component regulatory system gene baeR, efflux pump genes emrA/emrB and outer membrane coding gene ompA revealed that all mutant strains had less biofilm formation than the wild-type strain, which was further supported by the images from scanning electron microscopy and confocal laser scanning microscopy. The addition of amikacin, colistin, LL-37 or tannic acid decreased the biofilm formation ability of A. baumannii. In contrast, the addition of lower subinhibitory concentration tigecycline increased the biofilm formation ability of A. baumannii. Minimum biofilm eradication concentrations of amikacin, imipenem, colistin, and tigecycline were increased obviously for both wild type and multidrug resistant clinical strain A. baumannii VGH2. In conclusion, the biofilm formation ability of A. baumannii varied in different strains, involved many genes and could be influenced by many chemical compounds.

Influence of redox potential on leaching behavior of a solidified chromium contaminated soil.

Cement-based solidification/stabilization (S/S) technology is often used to remediate chromium (Cr) contaminated soils. The valence state and mobility of Cr in soils are closely related with redox potential (EH). However, Cr mobilization from the solidified soils influenced by EH has received little attention. In this study, semi-dynamic leaching tests and the toxicity characteristic leaching procedure (TCLP) were performed on a S/S treated Cr contaminated soil under various EH conditions. The effective diffusion coefficient and leachability index were obtained from the leaching data to investigate the leaching behavior of Cr from the S/S treated soil. Speciation of Cr remained in the sample after the leaching process was obtained through the sequential extraction procedures. The results show that an increase in EH increases the effective diffusion coefficient of Cr and, therefore, the amount of Cr leached. This result is attributed to immobile Cr(III) being oxidized to highly mobile Cr(VI). The leachability index results indicate that the cement solidification of Cr contaminated soil may not be appropriate under oxidizing conditions. For the TCLP and sequential extraction procedures, the leached amount of Cr exhibits a strong dependence on EH. As EH increases, the content of Cr remaining in the soil in unstable phases reduced, and more Cr was released to leachant.

Antimicrobial Susceptibility and Molecular Epidemiology of Proteus mirabilis Isolates from Three Hospitals in Northern Taiwan.

Of all the Proteus spp., Proteus mirabilis is the most common species identified in clinical specimens and is a leading agent of complicated urinary tract infection. This study was undertaken to understand the antimicrobial susceptibility, prevalence of antibiotic resistance genes, and molecular typing of P. mirabilis isolates collected from three hospitals in northern Taiwan. The results showed that the collected isolates of P. mirabilis were susceptible to most antibiotics except cefazolin and tigecycline. Many resistance genes were detected in the collected isolates, of which TEM genes were the most common. Resistance to third- or fourth-generation cephalosporins was related to the presence of at least one of the tested extended-spectrum ß-lactamase (ESBL) or AmpC genes. The presence of the VEB-1 gene seemed to be a good predictor for both cefepime and ceftazidime resistance, which was further supported by quantitative polymerase chain reaction results. Of the four imipenem-resistant P. mirabilis isolates, three isolates could hydrolyze imipenem by mass spectrometry analysis. Molecular typing by pulsed-field gel electrophoresis showed that the pulsotyping of the selected P. mirabilis isolates was heterogeneous. By analyzing the relationship of antimicrobial resistance and the presence of resistance genes, revision of the Clinical and Laboratory Standards Institute cefepime and ceftazidime MIC breakpoints for Enterobacteriaceae to predict ESBL producers might possibly be needed.

A method to assess influence of different medical tubing on biofilm formation by Acinetobacter baumannii.

In this study, we developed a method to assess influence of different medical tubing on biofilm formation by A. baumannii. The results of biofilm quantification and scanning electron microscopy showed that the biofilm formation susceptibility of different tubing materials was rubber latex > polyvinyl chloride > silicone.

Contribution of EmrAB efflux pumps to colistin resistance in Acinetobacter baumannii.

Efflux pumps play an important role in antimicrobial resistance for Acinetobacter baumannii. However, the function of the Emr pump system and the relationship between Emr and drug resistance has not been characterized in A. baumannii. In this study, four possible groups of emr-like genes were found by searching a genome database. Among them, A1S_1772 (emrB) and A1S_1773 (emrA) were demonstrated to be co-transcribed as a single operon. Moreover, during osmotic stress, A1S_1772 showed the largest change in gene expression compared to the other emrB-like genes, and deletion of A1S_1772 (AB ΔemrB) significantly slowed cell growth in 20% sucrose. Using a phenotypic microarray analysis, the AB ΔemrB mutant was more susceptible to colistin and nafcillin, paromomycin, spiramycin, and D,L-serine hydroxmate than the wild type. The spot assay, time kill assay and minimal inhibition concentration determination also indicated that the wild type could tolerate colistin better than the AB ΔemrB mutant. Finally, the increased expression levels of all emrB-like genes, including A1S_0775, A1S_0909, A1S_1772, and A1S_1799, in colistin resistance-induced A. baumannii further supported the possible involvement of the emrB genes in A. baumannii colistin resistance. Together, the Emr pump systems in A. baumannii contribute to adaptation to osmotic stress and resistance to colistin.

Distribution of different efflux pump genes in clinical isolates of multidrug-resistant Acinetobacter baumannii and their correlation with antimicrobial resistance.

BACKGROUND/PURPOSE: Efflux pumps are one of the major mechanisms of antimicrobial resistance in Acinetobacter baumannii. This study aimed to understand the distribution of different types of pump genes in clinical isolates of multidrug-resistant A. baumannii (MDRAB) and to reveal the relationship between their presence and expression with antimicrobial resistance. METHODS: MDRAB isolates were collected from five hospitals in Taiwan. Different categories of pump genes, including adeB, adeJ, macB, abeM, abeS, emrA-like, emrB-like, and craA, were chosen, and their presence in the collected isolates was determined. Three induced resistant strains of A. baumannii ATCC 17978 to tigecycline, imipenem, and amikacin were also included. The expressions of the selected pump genes were determined using quantitative reverse transcription-polymerase chain reaction. RESULTS: Twenty-one MDRAB clinical isolates were obtained from five hospitals. All of the studied pump genes were present in the collected MDRAB isolates except one isolate that lacked the emrA-like gene. The gene expression of these efflux pumps was variable among the strains. The upregulation of the adeB, adeJ, and macB genes was responsible for tigecycline resistance, and the increased abeS expression was strongly related to amikacin resistance. Of all the antibiotics studied, tigecycline was the strongest inducer of gene expression for many efflux pumps in A. baumannii. CONCLUSION: Efflux pump genes are universally present in the collected clinical MDRAB isolates. The upregulation of the adeB, adeJ, macB and abeS genes is more related with antibiotic resistance.

OmpA Binding Mediates the Effect of Antimicrobial Peptide LL-37 on Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii has recently emerged as an important pathogen in nosocomial infection; thus, effective antimicrobial regimens are urgently needed. Human antimicrobial peptides (AMPs) exhibit multiple functions and antimicrobial activities against bacteria and fungi and are proposed to be potential adjuvant therapeutic agents. This study examined the effect of the human cathelicidin-derived AMP LL-37 on A. baumannii and revealed the underlying mode of action. We found that LL-37 killed A. baumannii efficiently and reduced cell motility and adhesion. The bacteria-killing effect of LL-37 on A. baumannii was more efficient compared to other AMPs, including human ß-defensin 3 (hBD3) and histatin 5 (Hst5). Both flow cytometric analysis and immunofluorescence staining showed that LL-37 bound to A. baumannii cells. Moreover, far-western analysis demonstrated that LL-37 could bind to the A. baumannii OmpA (AbOmpA) protein. An ELISA assay indicated that biotin-labelled LL-37 (BA-LL37) bound to the AbOmpA74-84 peptide in a dose-dependent manner. Using BA-LL37 as a probe, the ~38 kDa OmpA signal was detected in the wild type but the ompA deletion strain did not show the protein, thereby validating the interaction. Finally, we found that the ompA deletion mutant was more sensitive to LL-37 and decreased cell adhesion by 32% compared to the wild type. However, ompA deletion mutant showed a greatly reduced adhesion defect after LL-37 treatment compared to the wild strain. Taken together, this study provides evidence that LL-37 affects A. baumannii through OmpA binding.

The Role of the Two-Component System BaeSR in Disposing Chemicals through Regulating Transporter Systems in Acinetobacter baumannii.

Bacterial two-component regulatory systems (TCSs) facilitate changes in gene expression in response to environmental stimuli. TCS BaeR regulons influence tigecycline susceptibility in Acinetobacter baumannii through positively regulating the pump genes adeA and adeB. In this study, we demonstrate that an additional two transport systems, AdeIJK and MacAB-TolC, are also regulated by BaeSR. In the wild type and clinical tigecycline-resistant A. baumannii strains, gene expression of AdeIJK and MacAB-TolC increased after tigecycline induction, implicating their importance to tigecycline resistance in addition to AdeABC. Phenotypic microarray results showed that A. baumannii is vulnerable to certain chemicals, especially tannic acid, after deleting baeR, which was confirmed using the spot assay. The wild-type strain of A. baumannii also exhibited 1.6-fold and 4.4-fold increase in gene expression of adeJ and macB in the medium with 100 µg/mL tannic acid, but the increase was fully inhibited by baeR deletion. An electrophoretic motility shift assay based on an interaction between His-BaeR and the adeA, adeI and macA promoter regions did not demonstrate direct binding. In conclusion, A. baumannii can use the TCS BaeSR in disposing chemicals, such as tannic acid and tigecycline, through regulating the efflux pumps.

Antimicrobial resistance in Acinetobacter baumannii: From bench to bedside.

Acinetobacter baumannii (A. baumannii) is undoubtedly one of the most successful pathogens in the modern healthcare system. With invasive procedures, antibiotic use and immunocompromised hosts increasing in recent years, A. baumannii has become endemic in hospitals due to its versatile genetic machinery, which allows it to quickly evolve resistance factors, and to its remarkable ability to tolerate harsh environments. Infections and outbreaks caused by multidrug-resistant A. baumannii (MDRAB) are prevalent and have been reported worldwide over the past twenty or more years. To address this problem effectively, knowledge of species identification, typing methods, clinical manifestations, risk factors, and virulence factors is essential. The global epidemiology of MDRAB is monitored by persistent surveillance programs. Because few effective antibiotics are available, clinicians often face serious challenges when treating patients with MDRAB. Therefore, a deep understanding of the resistance mechanisms used by MDRAB can shed light on two possible strategies to combat the dissemination of antimicrobial resistance: stringent infection control and antibiotic treatments, of which colistin-based combination therapy is the mainstream strategy. However, due to the current unsatisfying therapeutic outcomes, there is a great need to develop and evaluate the efficacy of new antibiotics and to understand the role of other potential alternatives, such as antimicrobial peptides, in the treatment of MDRAB infections.

Role of the BaeSR two-component system in the regulation of Acinetobacter baumannii adeAB genes and its correlation with tigecycline susceptibility.

BACKGROUND: Tigecycline resistance in Acinetobacter baumannii is primarily acquired through overexpression of the AdeABC efflux pump. Besides AdeRS, other two-component regulatory systems (TCSs) involving the regulation of this transporter have not been clarified. RESULTS: In this study, we found that the TCS genes baeR and baeS are co-transcribed and function as stress responders under high osmotic conditions. The baeSR and adeAB genes showed increased transcription in both the laboratory-induced and clinical tigecycline-resistant strains compared with the wild-type strain. The deletion of baeR in the ATCC 17978 strain led to 67-73% and 68% reduction in adeA and adeB expression, respectively, with a resultant 2-fold decrease in the tigecycline minimal inhibition concentration (MIC). In contrast, the overexpression of baeR resulted in a doubled tigecycline MIC, with a more than 2-fold increase in adeA and adeB expression. The influence of baeR knockout on adeAB gene expression can also be observed in the laboratory-induced tigecycline-resistant strain. A time-kill assay showed that the baeR deletion mutant showed an approximate 1-log10 reduction in colony forming units (CFUs) relative to the wild-type strain when the tigecycline concentration was 0.25 µg/mL throughout the assay period. The wild-type phenotype could be restored by trans-complementation with pWH1266-kanr-baeR. Increasing the tigecycline concentration to 0.5 µg/mL produced an even more marked 4.7-log10 reduction in CFUs of the baeR deletion mutant at 8 h, while only a 2.1-log10 reduction was observed for the wild-type strain. CONCLUSIONS: Taken together, these data show for the first time that the BaeSR TCS influences the tigecycline susceptibility of A. baumannii through the positive regulation of the resistance-nodulation-division efflux pump genes adeA and adeB.

Molecular epidemiology of integron-associated antimicrobial gene cassettes in the clinical isolates of Acinetobacter baumannii from northern Taiwan.

BACKGROUND: The aims of this study were to understand the molecular epidemiology of integron-associated gene cassettes in Acinetobacter baumannii across four hospitals in northern Taiwan and to clarify the relationship between the presence of integrons and antibiotic-resistant phenotypes. METHODS: Sixty-five A. baumannii isolates, collected from the patients of four regional hospitals in northern Taiwan in 2009, were tested for the presence of integrons and their associated gene cassettes. The susceptibility difference between integron-positive and integron-negative A. baumannii strains was analyzed. Antibiotic-resistant phenotypes among A. baumannii with different types of gene cassette array combinations were also compared. RESULTS: Around 72% of the A. baumannii isolates carried class 1 integrase genes. Despite this, only three gene cassette arrays were found in the integrons. Integron-positive strains were significantly more resistant to all the tested antibiotics than the integrase-negative strains. All the four types of A. baumannii with different gene cassette array combinations were multidrug-resistant in nature. Gene cassette array aacA4-catB8-aadA1 existed in all the integron-positive A. baumannii isolates. Repetitive-sequence-based PCR (rep-PCR) results revealed the prevalence of one major cluster of imipenem-resistant A. baumannii strains (84%) in the four regional hospitals. CONCLUSIONS: The presence of integrons with associated antimicrobial resistance gene cassettes can be used as a representative marker of multidrug resistance in A. baumannii. Some prevalent gene cassette arrays may exist among epidemiologically unrelated A. baumannii strains.

Clonal spread of multidrug-resistant Acinetobacter baumannii in eastern Taiwan.

BACKGROUND AND PURPOSE: This study was conducted to investigate the molecular epidemiology and antimicrobial susceptibility of multidrug-resistant (MDR) Acinetobacter baumannii to three types of antibiotics. METHODS: One hundred and thirty-four specimens of MDR A baumannii were collected from three branches (Taipei, Dalin, and Hualien branches) of Buddhist Tzu Chi Hospital, which are located in northern, southern, and eastern Taiwan, during 2007. Genotyping was performed by pulsed-field gel electrophoresis. Antibiotic susceptibilities to colistin, rifampicin, and tigecycline were determined. The synergistic effects of rifampin and colistin were also evaluated. RESULTS: Antibiotic susceptibility testing showed that 10.4%, 47.8% and 45.5% of the MDR A baumannii isolates are resistant to colistin, rifampicin, and tigecycline, respectively. A majority of the rifampicin-resistant isolates (62.7%) were found in the Haulien branch, whereas 62.2% of tigecycline-resistant isolates were found in the Taipei branch. The combination of colistin and rifampicin had a synergistic effect on all of the isolates. Genotyping by pulsed-field gel electrophoresis identified 17, 23, and 11 pulsotypes in the Taipei, Dalin, and Haulien branches, respectively. Furthermore, 74.5% of isolates in the Haulien branch were identified as one of three pulsotypes. Among 37 rifampicin-resistant and 22 tigecycline-resistant MDR A baumannii isolates found in the Haulien branch, 51.3% (19/37) and 50% (11/22) of the isolates belonged to the same clone, respectively. CONCLUSION: This study confirms the high prevalence of resistance to rifampicin and tigecycline in MDR A baumannii in the three hospitals that were studied, and the high proportion of identical strains that exist in eastern Taiwan.

Molecular epidemiology and antimicrobial resistance determinants of multidrug-resistant Acinetobacter baumannii in five proximal hospitals in Taiwan.

We investigated the molecular epidemiology, antibiotic susceptibility, and antimicrobial resistant gene determinants of 23 multidrug-resistant (MDR) Acinetobacter baumannii samples that were collected from 5 proximal hospitals in Taiwan during April and May 2009. Major antibiotic resistance varied from 82.6 to 100%. Fivw pulsotypes were observed to spread clonally among the 5 hospitals. PCR screening revealed high distributions of intI1 (91%), bla(OXA-23) (57%), bla(ampC) (100%), adeB (100%), adeJ (100%), and abeM (100%) genes, which were prevalent in the MDR A. baumannii isolates. Resistance gene expression was examined by reverse transcription-PCR, and showed that increased ampC expression was associated with ceftazidime resistance, but expression of adeB, adeJ, or abeM did not guarantee antimicrobial resistance phenotypes. In addition, imipenem resistance in some A. baumannii strains may be mainly modulated by genes other than bla(OXA-51-like). This is the first direct evidence indicating local spread of MDR A. baumannii in Taiwan. The resistance gene determinants are widely distributed in clonal and nonclonal-related isolates.

Emergence and dissemination of blaOXA-23-carrying imipenem-resistant Acinetobacter sp in a regional hospital in Taiwan.

BACKGROUND: The distribution and characterization of OXA-type carbapenemases in Acinetobacter sp in Taiwan has less been reported. The aim of the study was to investigate the molecular epidemiology and OXA-type carbapenemase genes in a regional hospital in Taiwan. METHODS: Imipenem-resistant Acinetobacter sp were collected between 2005 and 2007 in a regional hospital. Genotyping was performed by pulsed-field gel electrophoresis. OXA-type carbapenemase genes were determined by multiplex polymerase chain reaction (PCR) and gene sequencing. RESULTS: A total of 136 isolates were collected. Fifty-six pulsotypes were identified. None of the pulsotypes established predominance throughout the 3-year period. Multiplex PCR of blaOXA genes showed that 99% (135/136) of the Acinetobacter sp possessed blaOXA51-like genes. The coexistences of blaOXA51-like/blaOXA-23-like and blaOXA51-like/blaOXA-24-like were detected in 19% (26/136) and 1% (2/136) of the isolates, respectively. Among blaOXA-23-like gene-carrying isolates, two isolates (Pulsotypes 18 and 20) were found in 2006 and the remainder (n=24), including Pulsotypes 27 (n=18), 29 (n=1), 52 (n=3), and 53 (n=2), were found in 2007. Sequencing performed on the 26 representative isolates confirmed the presence of the blaOXA-23 carbapenemase gene. Analysis of the genetic content of blaOXA-23 showed that these genes were presumably chromosomal and associated with the upstream-located insertion sequence ISAba1. CONCLUSIONS: The emergence and imminent widespread of blaOXA-23-carrying imipenem-resistant Acinetobacter sp appeared in Taiwan during the period from 2006 to 2007.

Role of integrons in antimicrobial susceptibility patterns of Acinetobacter baumannii.

The relationship between the presence and types of integrons and the antimicrobial susceptibility patterns of Acinetobacter baumannii was investigated. A total of 134 non-duplicated A. baumannii isolates, 54.5% (n=73) of which were subsequently found to carry class 1 integrons, were collected from a regional hospital in Taiwan between March and September 2007. Only two types of gene cassette array, aacA4-catB8-aadA1 and aacC1-orfP-orfP-orfQ-aadA1, were identified. Susceptibility data showed that those strains carrying integrons were significantly more resistant to all antibiotics tested except ampicillin/sulbactam and imipenem. An epidemiological study revealed that the same integron could be found in different unrelated strains. These findings suggest that the presence of integrons in A. baumannii is responsible for both the horizontal transfer of antibiotic-resistance genes related to aminoglycosides and chloramphenicol and also represents a marker of multidrug resistance and epidemic potential.

Distribution of blaOXA-carrying imipenem-resistant Acinetobacter spp. in 3 hospitals in Taiwan.

We investigated the molecular epidemiology and OXA-type carbapenemase genes of 83 imipenem-resistant Acinetobacter spp. collected from 2 university hospitals (hospitals A and B) and a regional hospital (hospital C) during 2007 in Taiwan. Genotyping by pulsed-field gel electrophoresis identified 51 pulsotypes. None of the pulsotypes established predominance throughout the 3 hospitals. Multiplex polymerase chain reaction of blaOXA genes showed that 100% (18/18), 91%(31/34), and 100% (31/31) of the Acinetobacter spp. collected from hospital A, B, and C, respectively, possessed blaOXA-51-like genes. None of the strains carrying blaOXA-23-like and blaOXA-24-like genes were found in hospital A. The coexistences of blaOXA-51-like/blaOXA-23-like and blaOXA-51-like/blaOXA-24-like genes detected in hospitals B and C were 26% (9/34) and 12% (4/34) and 58% (18/31) and 3% (1/31), respectively. Among blaOXA-23-like gene-carrying isolates collected from hospitals, clonal spread of strains carrying the blaOXA-23 gene was detected in the regional hospital but not the other 2 university hospitals. The results suggest that interhospital dissemination of imipenem-resistant Acinetobacter spp. was not found in these hospitals. The increasing percentage of OXA-23 in OXA-type carbapenemases in Acinetobacter spp. from the regional hospitals to medical centers deserves further attention in Taiwan.

Clinical features and molecular epidemiology of multidrug-resistant Acinetobacter calcoaceticus-A baumannii complex in a regional teaching hospital in Taiwan.

We conducted a case-controlled study in a regional teaching hospital in Taiwan to investigate the clinical features and molecular epidemiology of multidrug-resistant Acinetobacter calcoaceticus-A baumannii (MDR Acb) complex. Case patients had higher mortality than controls did. MDR Acb complex acquisition risk factors include longer hospital stays, higher ratio of nasogastric tube and Foley catheter use, and more carbapenem use. All available isolates were divided into 36 subtypes by pulsed-field gel electrophoresis. The proportion of the same subtypes with their appearance within 1 and 2 months was 62.5% and 87.5%, respectively. We concluded that many different MDR Acb complex clones could be found in a hospital and that the same clones often spread on a small scale within a short period of time if no outbreaks noted.

Characterization of antimicrobial resistance patterns and integrons in human fecal Escherichia coli in Taiwan.

Two hundred and twenty-five fecal strains of Escherichia coli isolated from 109 non-hospitalized adults in 2006 were investigated for susceptibility to antibiotics and for the presence of integrons. High resistance rates in fecal strains of E. coli were observed for streptomycin (52.0%), ampicillin (50.2%), piperacillin (50.2%), trimethoprim/sulfamethoxazole (47.6%) and chloramphenicol (33.8%). Integrons were found in 31.5% (71/225) of the strains using an integrase gene PCR assay. Among 71 integrase-positive strains, 65 strains belonged to class 1 integrons, while the remainder belonged to class 2. Gene cassette patterns of class 1 integrons were further characterized by PCR and direct sequencing. Among those class 1 integrase-containing isolates, the integron cassette region was amplified by PCR in 40.0% (26 of 65) of isolates. Five different antimicrobial resistance gene cassette arrays were found in those isolates. These gene cassettes included those encoding resistance to trimethoprim (dfrV, dfrA7, dfrA12, dfrA17) and streptomycin (aadA1, aadA2, aadA5). Among those gene cassette arrays, dfrA12-orfF-aadA2 was found in 53.8% (14/26) of the isolates. These findings indicate that multidrug resistance of fecal flora is common in Taiwan and that integrons play an important role in resistance to trimethoprim and streptomycin in humans.

Favorable outcome of infective endocarditis due to Streptococcus agalactiae after conservative treatment.

Infective endocarditis due to Streptococcus agalactiae is uncommon and carries an ominous prognosis, leading some authors to advocate early surgery. This report describes an 83-year-old woman with community-acquired infective endocarditis due to S. agalactiae. The patient, who had a history of surgery for colon cancer, presented with fever, agitation and general malaise. She achieved a favorable outcome with antibiotic treatment only. For infective endocarditis due to S. agalactiae, appropriate antimicrobial agents should be started as soon as possible, with surgery reserved for those cases of particular indication.