Wen Liang Yu

Comparison of prevalence of virulence factors for Klebsiella pneumoniae liver abscesses between isolates with capsular K1/K2 and non-K1/K2 serotypes

Hypermucoviscosity, rmpA (regulator of mucoid phenotype), aerobactin (an iron siderophore), kfu (an iron uptake system), allS (associated with allantoin metabolism), and K1/K2 capsules are important virulence determinants in Klebsiella pneumoniae for liver abscesses. We determined the prevalence of these virulence factors of 50 nonrepeat K. pneumoniae isolates recovered from patients with primary liver abscesses who were treated at 2 medical centers in Taiwan. Virulence genes were surveyed by polymerase chain reaction analysis. The prevalence of hypermucoviscosity phenotype, plasmid-born rmpA, aerobactin, kfu, and allS genes revealed 96%, 100%, 100%, 100%, and 100% in 26 capsular K1 isolates; 90%, 100%, 100%, 0%, and 0% in 10 K2 isolates; and 79%, 86%, 93%, 50%, and 0% in 14 non-K1/K2 isolates; respectively. When injected into mice intraperitoneally, regardless of any capsule K serotype, K. pneumoniae isolates with hypermucoviscosity phenotype as well as presence of rmpA and aerobactin genes exhibited high virulence for mouse lethality (LD(50), <10(2) CFU). Without significant difference in the prevalence of expressing hypermucoviscosity phenotype and carriage of rmpA and aerobactin genes, these virulent non-K1/K2 isolates are as capable as K1/K2 isolates of causing primary liver abscesses.

In vitro efficacy of antimicrobial agents against high-inoculum or biofilm-embedded meticillin-resistant Staphylococcus aureus with vancomycin minimal inhibitory concentrations equal to 2 μg/mL (VA2-MRSA)

Minimal inhibitory concentration (MIC) creep in meticillin-resistant Staphylococcus aureus (MRSA) isolates has been observed in recent years. The potential roles of vancomycin-based combination regimens as well as linezolid and tigecycline against five clinical MRSA isolates with vancomycin MICs of 2 μg/mL (VA2-MRSA) were evaluated and compared in vitro. Antimicrobial susceptibility was studied by the agar dilution method. Anti-MRSA activities of linezolid, tigecycline, vancomycin, minocycline, rifampicin and fosfomycin alone as well as of three vancomycin-based combinations were studied by time-kill method and using a biofilm model. When VA2-MRSA at an inoculum of 1×10(5)colony-forming units (CFU)/mL was incubated with vancomycin, tigecycline, linezolid or rifampicin alone, bactericidal activity lasted for 48 h in time-kill analysis. At a higher inoculum of 1×10(7) CFU/mL, only linezolid demonstrated a bacteriostatic effect at 24 h and the inhibitory activity lasted for 36 h. However, bacterial growth was inhibited ≥2log(10) at 24 h and was even undetectable at 48 h with vancomycin plus fosfomycin or rifampicin. In biofilm studies, vancomycin plus fosfomycin or minocycline at susceptible breakpoint concentrations demonstrated an enhanced antibacterial effect comparable with linezolid and better than tigecycline. In conclusion, vancomycin plus fosfomycin or rifampicin exhibited a synergistic and better antibacterial effect than linezolid or tigecycline alone against high-inoculum planktonic VA2-MRSA. Vancomycin plus fosfomycin or minocycline compared with linezolid exhibited a similar inhibitory effect, better than tigecycline alone, against biofilm-embedded VA2-MRSA. Evaluating the toxicity and efficacy of high-dose vancomycin monotherapy for VA2-MRSA, the fosfomycin combination exhibited a rapid killing effect in both conditions and may provide another therapeutic choice.

In Vitro Efficacies and Resistance Profiles of Rifampin-Based Combination Regimens for Biofilm-Embedded Methicillin-Resistant Staphylococcus aureus

ABSTRACT To compare the in vitro antibacterial efficacies and resistance profiles of rifampin-based combinations against methicillin-resistant Staphylococcus aureus (MRSA) in a biofilm model, the antibacterial activities of vancomycin, teicoplanin, daptomycin, minocycline, linezolid, fusidic acid, fosfomycin, and tigecycline alone or in combination with rifampin against biofilm-embedded MRSA were measured. The rifampin-resistant mutation frequencies were evaluated. Of the rifampin-based combinations, rifampin enhances the antibacterial activities of and even synergizes with fusidic acid, tigecycline, and, to a lesser extent, linezolid, fosfomycin, and minocycline against biofilm-embedded MRSA. Such combinations with weaker rifampin resistance induction activities may provide a therapeutic advantage in MRSA biofilm-related infections.

Emergence of Two Klebsiella pneumoniae Isolates Harboring Plasmid-Mediated CTX-M-15 β-Lactamase in Taiwan

In recent years, cefotaximase (CTX-M)-type extended-spectrum β-lactamase-producing Enterobacteriaceae has been increasingly discovered worldwide (7). These β-lactamases confer a typical phenotype for which MICs of ceftriaxone and cefotaxime are higher than those of ceftazidime (7). In Taiwan, CTX-M-14 β-lactamase has been found in Klebsiella pneumoniae (9), and CTX-M-3 enzyme has been discovered in Escherichia coli (8), K. pneumoniae (9), and Serratia marcescens (11). In this report, we described the first isolation of Taiwanese CTX-M-15-producing K. pneumoniae isolates for which MICs of ceftazidime were high (≥128 μg/ml). Among 211 extended-spectrum β-lactamase-producing K. pneumoniae isolates collected from the whole country between January 1998 and June 2000, two isolates (KPN 154, a urine isolate from hospital N03, and KPN 176, a blood isolate from hospital N04) containing an uncharacterized enzyme with a pI of 8.8 were studied. Both clinical isolates of K. pneumoniae were susceptible to imipenem (MIC, 0.12 μg/ml) and meropenem (MICs, 0.064 to 0.094 μg/ml) but were resistant to cefotaxime (MIC, >256 μg/ml), ceftazidime (MIC, 128 to >256 μg/ml), and cefepime (MIC, 32 to 64 μg/ml), as determined by using the E-test (Table ​(Table1).1). Clavulanic acid totally restored the activities of cefotaxime, ceftazidime, and cefepime to levels at which the isolates were susceptible (MICs, 0.047 to 2 μg/ml). TABLE 1. Activity of various antimicrobial agentsa against two isolates of K. pneumoniae (KPN 154 and 176), two transconjugants (E154 and E176), and the recipient strain (E. coli J53-2) and their ribotypes, pIs, and bla gene characterization Plasmid DNA templates were prepared and purified for PCR. Amplification was performed using PCR primers for the bla genes encoding TEMs, SHVs, or CTX-M-3 β-lactamases (5, 9). Isoelectric focusing analysis revealed four enzymes with pIs of 5.4, 7.6, 8.2, and 8.8 in both isolates, which were matched to TEM-1, SHV-1, SHV-5, and CTX-M-15, respectively, by PCR and DNA sequence analysis (Table ​(Table11). Automated ribotyping using the RiboPrinter Microbial Characterization system (Qualicon, Inc., Wilmington, Del.) revealed nonclonal relatedness of both K. pneumoniae strains, but each belonged to two different major clones in Taiwan (KPN 154, ribotype 255.3; KPN 176, ribotype 691.5). Conjugation experiments provided evidence of transferable plasmids containing bla genes encoding SHV-5 (pI, 8.2) and/or CTX-M-15 (pI, 8.8). Both transconjugants revealed MIC profiles that were similar to those of the parent strains. The transconjugant E154 contained the gene blaCTX-M-15 without blaSHV-5, possibly indicating the failure of blaSHV-5 gene transference. Therefore, the presence of CTX-M-15 conferred to transconjugant E154 the ceftazidime resistance characterized in previous papers (1, 2, 6). This spectrum of hydrolysis has rarely been observed in other members of the CTX-M enzyme family (7). The mechanism of cefepime resistance may be the cumulative effects of CTX-M-15 and SHV-5 (as in KPN 154, KPN 176, and E176) or the presence of CTX-M-15 alone (as in E154). CTX-M-15, an Asp-240-Gly variant of CTX-M-3, increased the catalytic efficiency against ceftazidime (6). CTX-M-15 β-lactamase has already been identified in several countries, including India (2), Poland (1), Turkey (3), and the United Kingdom (4). Since CTX-M-15 differs from CTX-M-3 by only a single amino acid change and CTX-M-3 has also been widely disseminated among isolates of Enterobacteriaceae in Taiwan (8, 9, 11), it is not surprising that CTX-M-15 emerged in the Taiwan environment. Although not clonally related, these two emerging CTX-M-15-producing strains were documented to evolve from different major K. pneumoniae clones in Taiwan. Ribotype 691.5 has been recognized as an epidemic clone in northern Taiwan, and ribotype 255.3 has been recognized as another nationwide-epidemic clone (10). Although still rare, strains producing CTX-M-15 in Taiwan could potentially further disseminate, either clonally or by plasmid-related transmission.

In vitro synergistic antimicrobial effect of imipenem and colistin against an isolate of multidrug-resistant Enterobacter cloacae.

BACKGROUND/PURPOSE Enterobacter cloacae is an important nosocomial pathogen responsible for various infections. Little is known about the synergistic effects of imipenem and colistin against multidrug-resistant E. cloacae. Therefore, we investigated the in vitro effects of imipenem and colistin against a clinical isolate of multidrug-resistant E. cloacae. METHODS A strain of E. cloacae, designed Ent 831, was isolated from the sputum of a woman who developed severe pneumonia in a medical intensive care unit. Minimal inhibitory concentrations (MICs) of imipenem and colistin were determined by the agar dilution method. The synergistic effects were investigated using the time-kill method. RESULTS MICs of imipenem and colistin for E. cloacae strain Ent 831 were 0.5 microg/mL and 1.0 microg/mL, respectively. Using a standard inoculum (5 x 10(5)) CFU/mL), synergism was shown with a concentration of two times the MICs of imipenem and colistin. Furthermore, four times the MIC of imipenem completely inhibited bacterial growth for more than 48 hours, but four times the MICs of colistin resulted in re-growth after 4 hours. There was no synergism between imipenem and colistin at two times the MICs against a high concentration inoculum (6.24 x 10(6)) CFU/mL). Nevertheless, imipenem, with or without colistin, at a concentration of four times MICs could inhibit the growth of bacteria for more than 48 hours. CONCLUSION High-dose imipenem, alone or in combination with colistin, is effective against multidrug-resistant E. cloacae. Colistin alone, even at a high dose, is not effective. However, in vitro susceptibility to antimicrobial compounds does not always correlate with clinical success. Thus further testing of these antibiotic combinations in animal models is needed in order to predict their suitability for clinical use.

Screening Extended-spectrum β-Lactamase Production in Enterobacter cloacae and Serratia marcescens Using Antibiogram-based Methods

BACKGROUND/PURPOSE In Enterobacter cloacae and Serratia marcescens, AmpC beta-lactamases can confer resistance to the third-generation cephalosporins and oxacephems, but not to the fourth-generation cephalosporins. Extended-spectrum beta-lactamases (ESBL) may confer resistance to all extended-spectrum cephalosporins but not flomoxef. As difficult to detect, the ESBL phenotype of the intrinsically AmpC- producing E. cloacae and S. marcescens is not routinely screened in the clinical microbiology laboratories. The distinct antibiotic resistance phenotype between ESBL- and AmpC-producers may assist to differentiate the type of secreted beta-lactamases. Therefore, we attested the validity of an antibiogram-based method to predict the presence of ESBLs in both species. METHODS Polymerase chain reaction-based methods and antibiogram-based methods were compared for their detection of ESBL in 74 E. cloacae and 69 S. marcescens isolates recovered from patients hospitalized at two medical centers in Taiwan. Three major types of antibiogram were defined: type I (3s4s), susceptible to cefotaxime, ceftazidime, and cefepime; type II (3r4s), resistant to cefotaxime or ceftazidime, but susceptible to cefepime; and type III (3r4r), resistant to cefepime plus cefotaxime and/or ceftazidime. Furthermore, subtype-a and subtype-b were defined as being resistant and susceptible to flomoxef, respectively. RESULTS Overall, ESBL producers were identified in 20 (27.0%) of Enterobacter and 11 (15.9%) of Serratia isolates by polymerase chain reaction-based methods. All type I isolates of both species (n= 49) were non-ESBL producers. In E. cloacae, all subtype IIb (n = 6) and type III (n = 6) isolates produced ESBLs, but only 8 of 17 IIa isolates produced ESBLs. The IIb and III types had the highest positive predictive value (100%) and specificity (100%) for ESBL detection. In S. marcescens, type II isolates rarely produced ESBLs (4/57 isolates), while seven of type III (n = 8) isolates produced ESBLs. Type III antibiogram had the highest positive predictive value (87.5%) and specificity (98.3%) for ESBL detection. CONCLUSION The antibiograms of subtype IIb and type III are highly predictive for ESBL detection in E. cloacae, while type III is highly predictive for ESBL detection in S. marcescens. It is imperative to further examine ESBLs, focusing on the E. cloacae isolates with antibiogram subtype IIa.

Cytomegalovirus colitis in intensive care unit patients: Difficulties in clinical diagnosis☆ , ☆☆

PURPOSE Cytomegalovirus (CMV) infection occurs increasingly in critically ill patients in intensive care units (ICUs). We reported CMV colitis which has rarely been recognized in the ICU patients. METHODS CMV DNA was detected by polymerase chain reaction (PCR) for blood and/or stool samples. Definite diagnosis of CMV colitis required histopathology or CMV immunohistochemical staining of colorectal biopsies. We reviewed ICU patients characterized by positive blood or stool CMV-PCR with colorectal bleeding or water diarrhea. RESULTS We identified 18 patients (biopsy-proved, n=8; probable cases, n=10). The most common comorbidities were chronic renal disease, diabetes mellitus, and coronary artery disease. Stool CMV-PCR was positive in 7 of 10 patients (2 of 3 biopsy-proved and 5 of 7 probable cases). Colonoscopy was performed for 15 patients, revealing ulcerative or polypoid lesions. The endoscopists obtained colonic biopsies from 9 patients. Yet, the pathologists reported CMV colitis for 4 patients. Additional 4 patients were confirmed using immunohistochemical stain by the request of clinical physicians. Pseudomembranous colitis was found in 4 patients. CONCLUSION Diagnosis of CMV colitis seems difficult in clinical practice and need persistent communication between clinicians. The positive stool CMV-PCR result was a useful hint for adding immunohistochemical stain in mucosal biopsies to make a definite diagnosis of CMV colitis.

The outcomes of patients with severe dengue admitted to intensive care units

Abstract Outcomes of adult patients with dengue infections requiring intensive care unit (ICU) admissions remain unclear. We assessed the clinical manifestations and prognostic factors of patients critically ill with severe dengue. This retrospective study was done in a tertiary referral hospital with 96 adult ICU beds. All of the patients with laboratory-confirmed severe dengue infections and admitted to the ICU were enrolled between July 31 and November 31, 2015, during the large outbreak period. The medical records of all the recruited patients were reviewed for the following information: age, gender, clinical manifestations, disease severity scores, underlying conditions, laboratory examinations, and outcomes. The primary endpoint was to find the predictors of ICU mortality. During the study period, 4787 patients with dengue infections required ICU admission. One hundred forty-three (2.99%) were critically ill (mean age: 69.7 years). Hypertension (n = 90, 62.9%) and diabetes mellitus (n = 70, 49.0%) were the 2 most common underlying diseases. Eighty critically ill patients (55.9%) had cobacterial infections, and 33 had cobacteremia. The hematologic system failed most often, followed by thoracic and cardiovascular systems. Fever was the most common presentation (n = 112; 78.3%), followed by anorexia (n = 47; 32.9%) and abdominal pain (n = 46; 32.2%). Overall, 33 patients died (mortality rate: 23.1%). Multivariate analysis showed that ICU mortality was significantly associated with lower Glasgow Coma Scale (GCS) scores, lower platelet counts before ICU discharge, and more organ failures. The number of severe dengue patients who require ICU admission remains high. The mortality rate was associated with lower GCS scores, lower platelet counts, and more organ failures. In addition, more than half of the critically ill dengue patients had comorbid bacterial infections.

Low prevalence of rmpA and high tendency of rmpA mutation correspond to low virulence of extended spectrum β-lactamase-producing klebsiella pneumoniae isolates

Invasive syndrome caused by Klebsiella pneumoniae (KP), including liver abscess, is mainly caused by community-acquired strains with characteristics of positive hypermucoviscosity (HV) phenotype and regulator of mucoid phenotype A (rmpA) and transcriptional activator (rmpA2) genes. Extended- spectrum β-lactamase-producing KP (ESBL-KP) is commonly nosocomial and rarely HV-positive. We aimed to explore the reasons of the rarer prevalence of HV phenotype, rmpA and rmpA2 as well as the virulence phenotype among the ESBL-KP isolates from clinical specimens than those non-ESBL isolates. The β-lactamase genes, rmpA, rmpA2 and genes for K capsule serotype of 440 KP isolates were analyzed. The virulence of the isolates was characterized by the mouse lethality experiments. The prevalence rates of HV phenotype (∼50% vs. < 10%) as well as rmpA and rmpA2 genes (∼50–60% vs. < 20–30%) were significantly higher in non-ESBL group than in the ESBL group (p < 0.0001). Expression of HV phenotype in the rmpA-positive KP isolates was significantly rarer in the ESBL group than in non-ESBL group (33.3% vs. 91.9%, p < 0.0001). The frameshift mutations of rmpA and/or rmpA2 corresponded to negative HV phenotype of KP isolates that harbored the rmpA and/or rmpA2, resulting in variable mouse lethality (LD50, ∼103 - >5 × 107 CFU). The mutation rates might significantly differ among KP isolates from various sources. Virulence was dependent on rmpA-related HV phenotype. In conclusion, ESBL-KP isolates were less hypermucoviscous and less virulent than non-ESBL KP isolates, mostly due to concurrently lower carriage and higher mutation rates of the rmpA and rmpA2 genes.

In Vitro and In Vivo Intracellular Killing Effects of Tigecycline against Clinical Nontyphoid Salmonella Isolates Using Ceftriaxone as a Comparator

ABSTRACT Salmonella is an important, worldwide food-borne pathogen. Resistance to fluoroquinolones and cephalosporins has been increasingly reported, and new therapeutic agents are desperately needed. In this study, we evaluated the in vitro antimicrobial susceptibility of clinical nontyphoidal Salmonella isolates to tigecycline. Antibacterial activity of tigecycline, ceftriaxone, and ciprofloxacin were investigated by time-kill studies and the murine peritonitis model. The MIC50/MIC90 values of tigecycline, ceftriaxone, and ciprofloxacin against 76 Salmonella isolates were 0.25/0.5, 1/8, and 0.125/0.5 μg/ml, respectively. The intracellular inhibitory activity of tigecycline at 0.5 μg/ml (1× MIC) against Salmonella isolates in human peripheral blood mononuclear cells was sustained for 24 h. In a mouse peritonitis model, tigecycline reduced the extracellular and intracellular bacterial counts from 107 CFU/ml and 105 CFU/ml, respectively, to an undetectable level within 96 h. The results were similar to those obtained with ceftriaxone. The survival rate of mice exposed to tigecycline after being infected by an inoculum of 1 × 105 CFU was 80%, and that of mice exposed to ceftriaxone was 100%. When the inoculum was increased to 1.3 × 106 CFU, the survival rate of mice treated by tigecycline was 20%, and that of mice exposed to ceftriaxone was 0% (P = 0.2). When a ceftriaxone- and ciprofloxacin-resistant but tigecycline-susceptible isolate was tested, mice treated by tigecycline had a higher survival rate than those treated by ceftriaxone (15/20 [75%] versus 6/20 [30%]; P = 0.011). Our results suggest that tigecycline is at least as effective as ceftriaxone for murine Salmonella infections and warrants further clinical investigations to delineate its potential against human Salmonella infections.