Complete genome sequence of a newly isolated lytic bacteriophage,EFAP‐1 of Enterococcus faecalis,and antibacterial activity of its endolysin EFAL‐1

Aims: In this work, we aimed to identify an effective treatment of infections caused by Enterococcus spp. strains resistant to conventional antibiotics. Methods and Results: We report the isolation and characterization of a new lytic bacteriophage, designated bacteriophage EFAP‐1, that is capable of lysing Enterococcus faecalis bacteria that exhibit resistance to multiple antibiotics. EFAP‐1 has low sequence similarity to all known bacteriophages. Transmission electron microscopy confirmed that EFAP‐1 belongs to the Siphoviridae family. A putative lytic protein of EFAP‐1, endolysin EFAL‐1, is encoded in ORF 2 and was expressed in Escherichia coli. Recombinant EFAL‐1 had broad‐spectrum lytic activity against several Gram‐positive pathogens, including Ent. faecalis and Enterococcus faecium. Conclusions: The complete genome sequence of the newly isolated enterococcal lytic phage was analysed, and it was demonstrated that its recombinant endolysin had broad lytic activity against various Gram‐positive pathogens. Significance and Impact of the Study: Bacteriophage EFAP‐1 and its lytic protein, EFAL‐1, can be utilized as potent antimicrobial agents against Enterococcus spp. strains resistant to conventional antibiotics in hospital infections and also as environmental disinfectants to control disease‐causing Enterococcus spp. in dairy farms.     

Antibiotic resistance dynamics and isolation rate of staphylococci and enterococci from patients of reconstructive surgery units

The dynamics of isolation of staphylococci and enterococci from clinical material of patients and their antibiotic susceptibility within a 5-year period (2005-2009) was analysed. 5990 isolates were tested: 1250 isolates of Staphylococcus aureus, 3268 isolates of S. epidermidis, 1005 isolates of Enterococcus faecalis and 467 isolates of E. faecium. Grampositive infections were shown to be prevailing within the last 2-3 years, the nosocomial epidermal staphylococci more and more replacing S. aureus (the ratio of S. epidermidis and S. aureus in 2009 was 3.3). The isolation rate of E. faecalis significantly increased (by 3.5 times) and the ratio of E. faecalis and E. faecium in 2009 was 4.3. The microflora composition with respect to the isolation source was analysed and its clinical significance was estimated. The study of the antibiotic susceptibility showed that oxacillin had its own specific niche, while antibiotics active against resistant grampositive cocci, such as rifampicin, fusidic acid, fluoroquinolones (moxifloxacin), cefoxitin, as well as amoxicillin/clavulane in infections due to E. faecalis, might be considered as the drugs of choice. In the treatment of nosocomial infections, when the etiological role of MRSA or VRE is suspected or confirmed, the complex therapy should obligatory include the most active antibiotics (vancomycin or linezolid among them).     

外科危重病人呼吸道院内感染的调查分析

目的：了解外科危重病人呼吸道院内感染致病菌及其细菌耐药性情况．为临床防治提供依据。方法：对我院SICU1997年1月～1999年12月三年间从痰标本中所分离的致病菌及其细菌耐药性进行回顾性调查。结果：外科危重病人呼吸道内感染仍以G^-菌为主,占58．0％,其次真菌25．4％、G^ 菌16．6％,致病菌前四位分别铜绿假单胞菌、白色念珠菌、嗜麦芽窄食黄单胞菌和耐甲氧西林金黄色葡萄球菌。体外药物敏感试验显示主要的致病菌均呈多重耐药特性。结论：本SICU呼吸道院内感染的致病菌仍以G^-菌为主,致病菌呈多重耐药特性,掌握本科室呼吸道内感染致病菌谱及其耐药特性具有重要意义。     

下呼吸道感染患者病原菌分布及耐药性

目的分析下呼吸道感染患者病原菌分布及耐药性,指导临床合理用药。方法收集我院2013年至2015年院内感染患者痰标本进行细菌培养和药物敏感性试验。结果 3年共收集下呼吸道痰液标本21 615份,分离病原菌5 621株,阳性率为26.0%;其中革兰阴性(G~-)菌4 249株,占75.6%,以铜绿假单胞菌居多(20.7%);真菌764株,占13.6%,以白假丝酵母居多(12.6%);革兰阳性(G~+)菌608株,占10.8%,以金黄色葡萄球菌居多(9.9%)。药物敏感试验结果显示:G~-杆菌对亚胺培南、美洛培南耐药率最低,对青霉素类、喹诺酮类和部分三代头孢类等抗菌药耐药率较高(50.0%)。G~+球菌对万古霉素、利奈唑胺耐药率为零,对青霉素类、喹诺酮类和红霉素等抗菌药耐药率较高(40.0%)。结论下呼吸道感染患者病原菌以G~-杆菌为主,耐药性呈增长趋势,临床应加大病原菌分布检测及其耐药性监测力度,及时调整抗菌药物用药。     

Infectivity and resistance to antibiotics of bacterial strains isolated from patients hospitalised in intensive care units

The aim of this study was to evaluate the frequency of isolation and antimicrobial resistance testing of bacterial strains isolated from clinical specimens from patients hospitalized in three Intensive Care Units in Wroc?aw. The susceptibility of bacteria (107 strains) to selected antibiotics was determined. The results clearly show that non-fermentative rods were identified as the main agents causing pneumonia (58% of isolates). The second commonest pathogens were Gram-positive cocci (29%). The P. aeruginosa and E. cloacae strains were resistant to ampicillin, amoxicillin/clavulanate, cefuroxime and cefotaxime. All isolates of A. baumanii were susceptible only to imipenem. The rods of K. pneumoniae and E. coli were resistant to ampicillin, about 55% strains of both bacteria were sensitive to other antibiotics, except piperacillin/tazobactam, imipenem and ciprofloxacin. About 90% of methicillin resistant S. epidermidis strains were resistant to all antibiotics, except vancomycin (100% isolates were sensitive). ESBL were detected among E. cloace, K. pneumoniae and E. coli. We found P. aeruginosa rods producing MBL.     

674例新生儿败血症病原学及耐药性分析

目的:了解新生儿败血症病原学特点和致病菌耐药酶的产生与抗生素应用的相关性,为临床及时明确病原、正确选用抗生素提供依据。方法:对12年间新生儿科5 350例发热新生儿患儿无菌采集血液进行细菌培养,同时进行药敏分析和耐药酶检测,并了解采集标本前抗生素的应用情况。结果:5 350份血液标本中检出需氧菌674株,阳性率12.6%,其中革兰阴性杆菌155株,占23.0%,革兰阳性球菌504株,占74.8%。排在前八位的菌种是表皮葡萄球菌、溶血葡萄球菌、嗜麦芽假单胞菌、人葡萄球菌、肺炎克雷伯菌、大肠埃希菌、不动杆菌、粪肠球菌。革兰阳性球菌对青霉素,哌拉西林,苯唑西林耐药性高,对万古霉素100%敏感;革兰阴性杆菌对氨苄西林、哌拉西林,头孢呋辛耐药性高,对亚胺培南、头孢三代较敏感;感染产耐药酶菌患儿在检测前使用抗生素比率高于感染但未产耐药酶菌的患儿比率(X2=6.55,P<0.05),尤其第三代头孢菌素的应用,差异更显著(X2=12.17,P<0.005)。结论:新生儿败血症病原菌以表皮葡萄球菌和非发酵菌为主,但溶血葡萄球菌有上升趋势。加强各类标本的细菌学检测,预防败血症,减少耐药菌株的产生。     

Isolation and partial characterization of Salmonella Gallinarum bacteriophage

Infections caused by Salmonella remain a major public health problem worldwide. Animal food products, including poultry meat and eggs, are considered essential components in the individual’s daily nutrition. However, chicken continues to be the main reservoir for Salmonella spp.Poultry farmers use several types of antibiotics to treat pathogens. This can pose a health risk as pathogens can build antibiotic resistance in addition to the possibility of accumulation of these antibiotics in food products. The use of phages in treating poultry pathogens is increasing worldwide due to its potential use as an effective alternative to antibiotics. Phages have several advantages over antibiotics; phages are very specific to target bacteria, less chances of developing secondary infections, and they only replicate at the site of infection.Here we report the isolation of a bacteriophage from chicken feces. The isolated bacteriophage hosts on Salmonella Gallinarum, a common zoonotic infection that causes fowl typhoid, known to cause major losses to poultry sector. The isolated bacteriophage was partially characterized as a DNA virus resistant to RNase digestion with approximately 20 Kb genome. SDS-PAGE analysis of total viral proteins showed at least five major bands (21, 28, 42, 55 and 68 kDa), indicating that this virus is relatively small compared to other known poultry phages. The isolated bacteriophage has the potential to be an alternative to antibiotics and possibly reducing antibiotic resistance in poultry farms.     

Macrolide resistance and <Emphasis Type="Italic">In Vitro</Emphasis> selection of resistance to antibiotics in <Emphasis Type="Italic">Lactobacillus</Emphasis> isolates

Spreading of resistance to antibiotics is of great concern due to the increasing rate of isolation of multiresistant pathogens. Since commensal bacteria may transfer determinants of resistance to pathogens, studies on development of resistance should include also lactobacilli. Resistance to macrolides, penicillins and tetracycline was determined in 40 isolates of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus crispatus, and Lactobacillus casei isolated from faeces of apparently healthy volunteers. Frequency of mutation and changes in susceptibility after serial exposure to these antibiotics at concentrations of 4× and 8× MIC were evaluated in susceptible isolates. Acquired resistance was defined as an increment in MIC values of at least four times in respect to the pre-selection values. Resistance to macrolides and/or tetracycline was identified in 14 and 4 isolates, respectively. ermB gene and A2058G mutation in 23S rRNA were detected in macrolide resistant isolates. Frequencies of mutation of susceptible isolates (n=26) were lower for ampicillin and erythromycin than for tetracycline. Serial exposure to antibiotics led to selection of resistant mutants. However, acquired resistance was rather unstable and was lost after subcultures in antibiotic-free medium in most mutants. Resistance to erythromycin was associated to a A2058G mutation in 23S rRNA. In conclusion, results indicate that resistance to macrolides and tetracycline is present among intestinal lactobacilli. Decrease in susceptibility following serial exposure to antibiotics might occur in lactobacilli, in a strain- and antibiotic-dependent way. Since lactobacilli are often used as probiotics, their ability to acquire resistance should be evaluated for isolates candidate to be included in probiotics based products.     

Antibiotic resistance is prevalent in an isolated cave microbiome

Antibiotic resistance is a global challenge that impacts all pharmaceutically used antibiotics. The origin of the genes associated with this resistance is of significant importance to our understanding of the evolution and dissemination of antibiotic resistance in pathogens. A growing body of evidence implicates environmental organisms as reservoirs of these resistance genes; however, the role of anthropogenic use of antibiotics in the emergence of these genes is controversial. We report a screen of a sample of the culturable microbiome of Lechuguilla Cave, New Mexico, in a region of the cave that has been isolated for over 4 million years. We report that, like surface microbes, these bacteria were highly resistant to antibiotics; some strains were resistant to 14 different commercially available antibiotics. Resistance was detected to a wide range of structurally different antibiotics including daptomycin, an antibiotic of last resort in the treatment of drug resistant Gram-positive pathogens. Enzyme-mediated mechanisms of resistance were also discovered for natural and semi-synthetic macrolide antibiotics via glycosylation and through a kinase-mediated phosphorylation mechanism. Sequencing of the genome of one of the resistant bacteria identified a macrolide kinase encoding gene and characterization of its product revealed it to be related to a known family of kinases circulating in modern drug resistant pathogens. The implications of this study are significant to our understanding of the prevalence of resistance, even in microbiomes isolated from human use of antibiotics. This supports a growing understanding that antibiotic resistance is natural, ancient, and hard wired in the microbial pangenome.     

Infectivityand resistance to antibiotics of non-fermentative rods isolated from patients hospitalised in pediatric center of Lower Silesia in Wrocław in the years 2000-2006

The aim of this study was to evaluate the frequency of isolation and antimicrobial resistance testing of non-fermentative rods isolated from clinical specimens from patients hospitalized in Korczak Pediatric Center of Lower Silesia in Wroc?aw. The susceptibility of bacteria to selected antibiotics was determined. The commonest pathogens were Pseudomonas rods (81.8%) isolated from respiratory system and urine of patients hospitalized in unit intensive care. Variety of resistance patterns were detected in bacteria. ESBL were detected the most of S. maltophilia. Strains of Pseudomonas and Acinetobacter resistant to carbapenems were detected with a frequency of 5.5% and 35.9%.     

Antibiotics and the resistant microbiome

Since the discovery and clinical application of antibiotics, pathogens and the human microbiota have faced a near continuous exposure to these selective agents. A well-established consequence of this exposure is the evolution of multidrug-resistant pathogens, which can become virtually untreatable. Less appreciated are the concomitant changes in the human microbiome in response to these assaults and their contribution to clinical resistance problems. Studies have shown that pervasive changes to the human microbiota result from antibiotic treatment and that resistant strains can persist for years. Additionally, culture-independent functional characterization of the resistance genes from the microbiome has demonstrated a close evolutionary relationship between resistance genes in the microbiome and in pathogens. Application of these techniques and novel cultivation methods are expected to significantly expand our understanding of the interplay between antibiotics and the microbiome.     

Combination drugs, an emerging option for antibacterial therapy

The emerging and sustained resistance to antibiotics and the poor pipeline of new antibacterials is creating a major health issue worldwide. Bacterial pathogens are increasingly becoming resistant even to the most recently approved antibiotics. Few antibiotics are being approved by regulatory organizations, which reflects both the difficulty of developing such agents and the fact that antibiotic discovery programs have been terminated at several major pharmaceutical companies in the past decade. As a result, the output of the drug pipelines is simply not well positioned to control the growing army of resistant pathogens, although academic institutions and smaller companies are trying to fill that gap. An emerging option to fight such pathogens is combination therapy. Combinations of two antibiotics or antibiotics with adjuvants are emerging as a promising therapeutic approach. This article provides and discusses clinical and scientific challenges to support the development of combination therapy to treat bacterial infections.     

Occurrence and susceptibility to antibiotics of carbapenem-resistant Pseudomonas aeruginosa strains between 1998 and 2009

P. aeruginosa rods are dangerous pathogens mainly responsible for nosocomial infections of different localization. Resistance to carbapenems, observed among them, is a serious threat due to ability to be transmitted between bacterial species. The aim of our study was to retrospectively evaluate the frequency of isolation and susceptibility to antibiotics of imipenem- and meropenem-resistant P. aeruginosa strains isolated between 1998 and 2009 from patients of University Hospital No 1 of dr A. Jurasz in Bydgoszcz. Study shows increasing number of isolation that type of strains from 19 in 1998 to 144 in 2009. Among all isolated P. aeruginosa strains majority was obtained from patients of the Intensive Care Units, Rehabilitation and Surgery Clinics. Examined strains of P. aeruginosa rods were mainly isolated from urine (20.5%), bronchoalveolar lavage (17.7%) and wound swabs (14.5%) samples. The isolates demonstrated frequently resistance to carbenicillin (> or 66.7%), ticarcillin (> or = 72.7%) and cefotaxime (> or = 75.6%). The lowest rate of resistant strains was observed in case of ceftazidime (< or = 68.8%), aztreonam (< or = 47.4%) and colistin (< or = 1.7%) suggesting the highest activity of that antimicrobials against infections caused by examined strains.     

Antibiotic Resistance Determinants in a Pseudomonas putida Strain Isolated from a Hospital

Environmental microbes harbor an enormous pool of antibiotic and biocide resistance genes that can impact the resistance profiles of animal and human pathogens via horizontal gene transfer. Pseudomonas putida strains are ubiquitous in soil and water but have been seldom isolated from humans. We have established a collection of P. putida strains isolated from in-patients in different hospitals in France. One of the isolated strains (HB3267) kills insects and is resistant to the majority of the antibiotics used in laboratories and hospitals, including aminoglycosides, ß-lactams, cationic peptides, chromoprotein enediyne antibiotics, dihydrofolate reductase inhibitors, fluoroquinolones and quinolones, glycopeptide antibiotics, macrolides, polyketides and sulfonamides. Similar to other P. putida clinical isolates the strain was sensitive to amikacin. To shed light on the broad pattern of antibiotic resistance, which is rarely found in clinical isolates of this species, the genome of this strain was sequenced and analysed. The study revealed that the determinants of multiple resistance are both chromosomally-borne as well as located on the pPC9 plasmid. Further analysis indicated that pPC9 has recruited antibiotic and biocide resistance genes from environmental microorganisms as well as from opportunistic and true human pathogens. The pPC9 plasmid is not self-transmissible, but can be mobilized by other bacterial plasmids making it capable of spreading antibiotic resistant determinants to new hosts.     

Resistance of Shigella to antibiotics

Antibiotic sensitivity of 104 Shigella clinical strains and 104 Escherichia coli strains isolated from patients with acute dysentery not treated with antibiotics in 1986-1987 was studied. It was shown that 100 per cent of the dysentery pathogens and colon bacilli were antibiotic resistant. Strains resistant simultaneously to chloramphenicol, ampicillin, streptomycin, tetracycline, monomycin and kanamycin were the most frequent among the dysentery pathogens. Colon bacilli and dysentery pathogens isolated from the same patient had specific sets of antibiotic resistance markers. Pathogenetic therapy of dysentery and exclusion of antibiotic use for several years did not result in lower numbers of Shigella antibiotic resistant strains.     

伤口分泌物的病原菌分布及耐药性分析

目的调查福建省龙岩市第二医院伤口分泌物病原菌的分布及耐药情况,为合理应用抗生素提供依据。方法收集2012年1月至2013年5月患者伤口分泌物标本,采用常规方法进行分离培养,用VITEK-2 Compact全自动微生物分析仪系统进行鉴定及药敏分析。结果送检503份标本,培养阳性272份,阳性率为54. 1% ;病原菌检出343株,其中革兰阴性菌189株占55. 1%,革兰阳性菌151株占44.0%,真菌3株占0.9% ;前5位的病原菌分别为金黄色葡萄球菌、凝固酶阴性葡萄球菌、铜绿假单胞菌、大肠埃希菌、鲍曼不动杆菌。耐甲氧西林金黄色葡萄球菌（MRSA）和耐甲氧西林凝固酶阴性葡萄球菌（MRCNS）的检出率分别是30.4%、82. 1%。粪肠球菌中耐高浓度氨基糖苷类肠球菌（HLAR）的检出率为55%。革兰阳性菌对万古霉素、替加环素、利奈唑胺未出现耐药菌株。铜绿假单胞菌、肠杆菌科细菌对亚胺培南的耐药率分别为5. 6%、0。大肠埃希菌中超广谱P-内酰胺酶（ESBL）的检出率是42.9%。鲍曼不动杆菌对头孢哌酮/舒巴坦的耐药率最低为25.0%,对其他抗菌药物耐药严重,多数抗菌药物的耐药率均〉45%。结论伤口感染的主要病原菌是革兰阴性菌,多重耐药菌株比例较高,临床应根据药敏结果合理选用抗生素,减少新的耐药菌株出现。     

肺结核合并非发酵菌下呼吸道感染的临床特征及耐药性分析

目的了解肺结核患者合并非发酵菌下呼吸道感染的临床特征及耐药性,为临床合理使用抗菌药物提供依据。方法采用ATB全自动细菌鉴定仪,对临床分离菌株进行菌种鉴定,用K．B法对非发酵菌做药物敏感试验。结果从肺结核患者下呼吸道标本中共分离非发酵菌156株,其中铜绿假单胞菌最多,占46．5％,其次为鲍曼不动杆菌和嗜麦芽寡养单胞菌,分别占37．8％和9．6％。药敏试验显示5种非发酵菌对多种抗生素均表现为高度耐药或多重耐药,高于相关研究,差异有统计学意义（P≤0．05）。结论肺结核患者肺部感染非发酵菌的分离率较高,多重耐药现象严重,临床应重视非发酵菌感染和耐药性监测。     

Prevalence of antibiotic resistance profile in relation to phylogenetic background among commensal Escherichia coli derived from various mammals.

The paper describes the prevalence of resistant strains within the genetic structure of E. coli (phylogenetic group A, B1, B2 and D). A total of 200 commensal E. coli strains have been derived from 10 species of healthy animals residing on ZOO Safari Park area, in Swierkocin, Poland. The phylogenetic structure of E. coli has been analysed with the use of a PCR-based method. The strains were tested in terms of their susceptibility to eight classes of antibiotics: aminoglycosides, penicillins, cephalosporins, tetracyclines, nitrofurans, sulphonamides, phinicols, and quinolones. The genetic structure of E. coli revealed a not uniform distribution of strains among the four phylogenetic groups with significantly numerous representation of groups A and B1. Resistant E. coli were found within each of the phylogenetic groups. Strains resistant to one class of antibiotics occurred significantly more frequently in phylogenetic groups B2 and D (potential pathogens), whereas strains resistant to more than one class of antibiotics belonged to phylogenetic groups A and B1 (typical commensals) in a prevailing number of cases.     

Antimicrobials, drug discovery, and genome mining

Over the years, antibiotics have provided an effective treatment for a number of microbial diseases. However recently, there has been an increase in resistant microorganisms that have adapted to our current antibiotics. One of the most dangerous pathogens is methicillin-resistant Staphylococcus aureus (MRSA). With the rise in the cases of MRSA and other resistant pathogens such as vancomycin-resistant Staphylococcus aureus, the need for new antibiotics increases every day. Many challenges face the discovery and development of new antibiotics, making it difficult for these new drugs to reach the market, especially since many of the pharmaceutical companies have stopped searching for antibiotics. With the advent of genome sequencing, new antibiotics are being found by the techniques of genome mining, offering hope for the future.     

Diversity of antifungal and plant-associated Serratia plymuthica strains

A total of 21 plant-associated Serratia plymuthica strains were characterized phenotypically by their nutritional patterns, susceptibility to antibiotics, antifungal and haemolytic properties, and genotypically by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA, PCR fingerprints using BOX primers (BOX-PCR) and pulsed-field gel electrophoresis (PFGE) after digestion with SpeI. All of the investigated strains demonstrated antifungal activity in vitro against fungal pathogens while only six strains produced the antifungal antibiotic prodigiosin. Haemolytic activity and antibiotic resistance patterns were investigated to assess the risk associated with the use of isolates in plant protection. The strains were haemolytic at human-relevant temperatures. The level of resistance to antibiotics was low. This work shows that BOX-PCR and PFGE are useful fingerprinting methods to characterize Ser. plymuthica strains, although the discriminatory effect between the two methods differed. Computer-assisted analysis of phenotypic and genotypic features demonstrated relationships between the origin of isolation, the production of prodigiosin and the molecular fingerprint.