Yali Gong

Antibacterial properties of Acinetobacter baumannii phage Abp1 endolysin (PlyAB1)

BackgroundAcinetobacter baumannii has emerged as one of the most important hospital-acquired pathogens in the world, because of its resistance to almost all available antibiotic drugs. Endolysins from phages are attracting increasing interest as potential antimicrobial agents, especially for drug-resistant bacteria. We previously isolated and characterized Abp1, a virulent phage targeting the multidrug-resistant A. baumannii strain, AB1.MethodsTo evaluate the antimicrobial potential of endolysin from the Abp1 phage, the endolysin gene plyAB1 was cloned and over-expressed in Escherichia coli, and the lytic activity of the recombinant protein (PlyAB1) was tested by turbidity assessment and bacteria counting assays.ResultsPlyAB1 exhibits a marked lytic activity against A. baumannii AB1, as shown by a decrease in the number of live bacteria following treatment with the enzyme. Moreover, PlyAB1 displayed a highly specific lytic effect against all of the 48 hospital-derived pandrug-resistant A. baumannii isolates that were tested. These isolates were shown to belong to different ST clones by multilocus sequence typing.ConclusionsThe results presented here show that PlyAB1 has potential as an antibiotic against drug-resistant A. baumannii.

The chromosomal SezAT toxin–antitoxin system promotes the maintenance of the SsPI‐1 pathogenicity island in epidemic Streptococcus suis

Streptococcus suis has emerged as a causative agent of human meningitis and streptococcal toxic shock syndrome over the last years. The high pathogenicity of S. suis may be due in part to a laterally acquired pathogenicity island (renamed SsPI‐1), which can spontaneously excise and transfer to recipients. Cells harboring excised SsPI‐1 can potentially lose this island if cell division occurs prior to its reintegration; however, attempts to cure SsPI‐1 from the host cells have been unsuccessful. Here, we report that an SsPI‐1‐borne Epsilon/Zeta toxin–antitoxin system (designated SezAT) promotes SsPI‐1 stability in bacterial populations. The sezAT locus consists of two closely linked sezT and sezA genes encoding a toxin and its cognate antitoxin, respectively. Overproduction of SezT induces a bactericidal effect that can be neutralized by co‐expression of SezA, but not by its later action. When devoid of a functional SezAT system, large‐scale deletion of SsPI‐1 is straightforward. Thus, SezAT serves to ensure inheritance of SsPI‐1 during cell division, which may explain the persistence of epidemic S. suis. This report presents the first functional characterization of TA loci in S. suis, and the first biochemical evidence for the adaptive significance of the Epsilon/Zeta system in the evolution of pathogen virulence.

Multilocus Sequence Typing Analysis of Carbapenem-Resistant Acinetobacter baumannii in a Chinese Burns Institute

Acinetobacter baumannii is a leading pathogen responsible for nosocomial infections. The emergence of carbapenem-resistant A. baumannii (CRAB) has left few effective antibiotics for clinicians to use. To investigate the temporal evolutionary relationships among CRAB strains, we collected 248 CRAB isolates from a Chinese burns institute over 3 years. The prevalence of the OXA-23 gene was detected by polymerase chain reaction. Multilocus sequence typing was used to type the CRAB strains and eBURST was used to analyze their evolutionary relationships. Wound surfaces (41%), sputa (24%), catheters (15%), and bloods (14%) were the four dominant isolation sources. Except for minocycline (33.5%) and sulbactam/cefoperazone (74.6%), these CRAB strains showed high resistance rates (>90%) to 16 tested antibiotics. The 248 isolates fall into 26 sequence types (STs), including nine known STs and 17 unknown STs. The majority (230/248) of these isolates belong to clonal complex 92 (CC92), including eight isolates belonging to seven unreported STs. A new CC containing 11 isolates grouped into four new STs was identified. The OXA-23 gene was detected at high prevalence among the CRAB isolates and the prevalence rate among the various STs differed. The majority of the isolates displayed a close evolutionary relationship, suggesting that serious nosocomial spreading and nosocomial infections of CRAB have occurred in the burn unit. In conclusion, the main CC for CRAB in this Chinese burn unit remained unchanged during the 3-year study period, and a new CC was identified. CC92 was the dominant complex, and more attention should be directed toward monitoring the new CC we have identified herein.

Burn Serum Increases Staphylococcus aureus Biofilm Formation via Oxidative Stress

Staphylococcus aureus is a common pathogen isolated from burn patients that can form biofilms on burn wounds and implanted deep vein catheters, which often leads to refractory infections or even biofilm-related sepsis. As biofilm formation is usually regulated by environmental conditions, we hypothesized that serum composition may be altered after burn injury, potentially affecting the ability of infecting bacteria to form biofilms. As predicted, we observed that serum from burn-injured rats increases biofilm formation by S. aureus and also induces bacterial aggregation and adherence to human fibronectin and fibrinogen. Analysis of potential regulatory factors revealed that exposure to burn serum decreases expression of the quorum-sensing agr system and increases mRNA levels of some biofilm inducers such as sarA and icaA. In addition, we also observed that burn serum imposes oxidative stress and increases expression of key oxidoreductase genes (sodA, sodM, katA, and ahpC) in S. aureus. Importantly, the ability of burn serum to enhance biofilm formation and bacterial cell aggregation can be abrogated by treatment with an antioxidant. Taken together, these findings indicate that burn serum increases S. aureus biofilm formation via elevated oxidative stress, and may lead to novel strategies to control biofilm formation and infection in burn patients.

A 5-year Survey Reveals Increased Susceptibility to Glycopeptides for Methicillin-Resistant Staphylococcus aureus Isolates from Staphylococcus aureus Bacteremia Patients in a Chinese Burn Center

Methicillin-resistant Staphylococcus aureus (MRSA) infections are prevalent in burn wards, and are especially serious in S. aureus bacteremia (SAB) patients. Glycopeptides and daptomycin are effective against MRSA infections, but MIC creeps can reduce their efficacy. Our object was to perform a molecular epidemiological investigation of S. aureus isolates in our burn center and to evaluate MICs for antimicrobials against SAB-associated MRSA isolates. A total of 259 S. aureus isolates, obtained from August 2011 to July 2016, were used in this study. Multiple molecular typing was used for molecular epidemiological analysis. E-tests were used to determine MICs of vancomycin, teicoplanin, and daptomycin for SAB-associated MRSA isolates. MIC values were stratified by collection date or source and compared. Spearmans test was used to analyze MICs correlations amongst tested antimicrobials. ST239-MRSA-III-t030-agrI clone was found to be dominant in both SAB and non-SAB patients, and significantly more in SAB patients (P < 0.0001). SAB-MRSA isolates exhibited decreased MICs for vancomycin, teicoplanin, and daptomycin during the 5-year period. Compared to those isolated from catheters or wounds, SAB-MRSA isolates from the bloodstream were less susceptible to vancomycin and daptomycin, but more susceptible to teicoplanin. MICs Correlation was found only between vancomycin and daptomycin in MRSA isolates from the bloodstream (rho = 0.250, P = 0.024). In conclusion, our results suggest that MRSA infections are still serious problems in burn centers. In contrast to most other studies, we observed increased susceptibility to glycopeptides and daptomycin against SAB-associated MRSA in our center from 2011 to 2016, suggesting the use of glycopeptides does not lead to MIC creeps. Isolates from different sites of the body may exhibit different levels of susceptibility and change trend over time for different antimicrobials, antimicrobials selection for MRSA infections should be considered comprehensively.

Epidemiology and resistance features of Acinetobacter baumannii isolates from the ward environment and patients in the burn ICU of a Chinese hospital.

Acinetobacter baumannii is an important opportunistic pathogen that causes severe nosocomial infections, especially in intensive care units (ICUs). Over the past decades, an everincreasing number of hospital outbreaks caused by A. baumannii have been reported worldwide. However, little attention has been directed toward the relationship between A. baumannii isolates from the ward environment and patients in the burn ICU. In this study, 88 A. baumannii isolates (26 from the ward environment and 62 from patients) were collected from the burn ICU of the Southwest Hospital in Chongqing, China, from July through December 2013. Antimicrobial susceptibility testing results showed that drug resistance was more severe in isolates from patients than from the ward environment, with all of the patient isolates being fully resistant to 10 out of 19 antimicrobials tested. Isolations from both the ward environment and patients possessed the β-lactamase genes blaOXA-51, blaOXA-23, blaAmpC, blaVIM, and blaPER. Using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST), these isolates could be clustered into 4 major PFGE types and 4 main sequence types (ST368, ST369, ST195, and ST191) among which, ST368 was the dominant genotype. Epidemiologic and molecular typing data also revealed that a small-scale outbreak of A. baumannii infection was underway in the burn ICU of our hospital during the sampling period. These results suggest that dissemination of β-lactamase genes in the burn ICU might be closely associated with the high-level resistance of A. baumannii, and the ICU environment places these patients at a high risk for nosocomial infection. Cross-contamination should be an important concern in clinical activities to reduce hospitalacquired infections caused by A. baumannii.

Risk factors for central line-associated bloodstream infection in patients with major burns and the efficacy of the topical application of mupirocin at the central venous catheter exit site

AIM The aim of this study was to evaluate the efficacy of the topical administration of mupirocin and other practices in central venous catheter (CVC) care to prevent central line-associated bloodstream infections (CLABSI) in patients with major burns. METHODS Patients with major burns admitted to a burn ICU were divided into four groups and disinfected at the CVC exit site with single povidone iodine (PVP-I) or PVP-I plus topical mupirocin ointment three times a day or once a day, respectively. The bacterial colonization of the skin at the CVC exit site and CVC tips and the incidence of CLABSI were recorded, and the risk factors were analyzed. RESULTS Administering mupirocin (RR=0.316, p=0.001), increasing the frequency of insertion-site care (RR=0.604, p=0.008), and avoiding cannulation at the burn site (RR=0.148, p<0.001) reduced skin colonization at the CVC insertion site. Topical administration of mupirocin significantly reduces both the bacterial colonization rate at CVC tips (RR=0.316, p=0.001) and the incidence of CLABSI (5.3 vs. 29.1 per 1000 catheter days, p<0.001). CONCLUSIONS Mupirocin is effective in the prophylaxis of CLABSI. Other CVC care practices were also found to affect the level of bacterial colonization, but their efficacy in preventing CLABSI needs to be evaluated further.

Panton-Valentine Leucocidin (PVL) as a Potential Indicator for Prevalence, Duration, and Severity of Staphylococcus aureus Osteomyelitis

Staphylococcus aureus is the most common cause of the difficult-to-treat osteomyelitis (OM). To better diagnose and manage S. aureus OM, especially for severe and long duration cases, indicators for risk prediction and severity evaluation are needed. Here, 139 clinical S. aureus isolates from orthopedic infections were divided into OM group (60 isolates from 60 OM patients) and non-OM group (79 isolates from 79 non-OM patients). Molecular types, antimicrobial susceptibility, and virulence factor profiles were evaluated and compared between the two groups to identify potential indicators associated with the prevalence of S. aureus OM. Clinical manifestations and laboratory data were analyzed to identify indicators affecting OM duration and severity. We found that some sequence types were specific to OM infection. The pvl, bbp, and ebps genes were associated with S. aureus OM prevalence. The pvl, bbp, and sei genes were associated with relatively longer OM duration. Panton-Valentine leucocidin (PVL)-positive S. aureus OM presented more serious inflammatory responses. Our results emphasize the significance of PVL in affecting the prevalence, duration, and severity of S. aureus OM. Diagnosing and monitoring PVL-related S. aureus OM may help direct better prognosis and treatment of these patients.

The Interaction of N-Acetylcysteine and Serum Transferrin Promotes Bacterial Biofilm Formation

Background/Aims: N-acetylcysteine (NAC) is a novel and promising agent with activity against bacterial biofilms. Human serum also inhibits biofilm formation by some bacteria. We tested whether the combination of NAC and human serum offers greater anti-biofilm activity than either agent alone. Methods: Microtiter plate assays and confocal laser scanning microscopy were used to evaluate bacterial biofilm formation in the presence of NAC and human serum. qPCR was used to examine expression of selected biofilm-associated genes. Extracellular matrix (ECM) was observed by transmission electron microscopy. The antioxidants GSH or ascorbic acid were used to replace NAC, and human transferrin, lactoferrin, or bovine serum albumin were used to replace serum proteins in biofilm formation assays. A rat central venous catheter model was developed to evaluate the effect of NAC on biofilm formation in vivo. Results: NAC and serum together increased biofilm formation by seven different bacterial strains. In Staphylococcus aureus, expression of genes for some global regulators and for genes in the ica-dependent pathway increased markedly. In Pseudomonas aeruginosa, transcription of las, the PQS quorum sensing (QS) systems, and the two-component system GacS/GacA increased significantly. ECM production by S. aureus and P. aeruginosa was also enhanced. The potentiation of biofilm formation is due mainly to interaction between NAC and transferrin. Intravenous administration of NAC increased colonization by S. aureus and P. aeruginosa on implanted catheters. Conclusions: NAC used intravenously or in the presence of blood increases bacterial biofilm formation rather than inhibits it.

Characterization and interstrain transfer of prophage pp3 of Pseudomonas aeruginosa

Prophages are major contributors to horizontal gene transfer and drive the evolution and diversification of bacteria. Here, we describe the characterization of a prophage element designated pp3 in the clinical Pseudomonas aeruginosa isolate PA1. pp3 spontaneously excises from the PA1 genome and circularizes at a very high frequency of 25%. pp3 is likely to be a defective prophage due to its inability to form plaques on P. aeruginosa indicator strains, and no phage particles could be detected in PA1 supernatants. The pp3-encoded integrase is essential for excision by mediating site-specific recombination at the 26-bp attachment sequence. Using a filter mating experiment, we demonstrated that pp3 can transfer into P. aeruginosa recipient strains that do not possess this element naturally. Upon transfer, pp3 integrates into the same attachment site as in PA1 and maintains the ability to excise and circularize. Furthermore, pp3 significantly promotes biofilm formation in the recipient. Sequence alignment reveals that the 26-bp attachment site recognized by pp3 is conserved in all P. aeruginosa strains sequenced to date, making it possible that pp3 could be extensively disseminated in P. aeruginosa. This work improves our understanding of the ways in which prophages influence bacterial behavior and evolution.