The spread of Klebsiella in obstetrical hospitals and the biological properties of the isolated strains]

Examination of 278 newborn infants, parturients, and medical personnel in two maternity hospitals revealed a high level of Klebsiella colonization of all examined biotopes of infants (the nasal cavity in up to 36.5% of cases) and the skin of the mammary glands of nursing mothers (in 36.2% of cases). In the intestine and the nasal cavity of parturient women and medical personnel Klebsiella could be detected 3-10 times more often than in the same biotopes of nonhospitalized pregnant women. From 254 objects of the hospital environment Klebsiella were isolated in 9.05% of cases. The possibility of the transmission of Klebsiella in hospitals by patients with inflammatory processes in their genitals were established. The diversity of the serological picture of strains of most K-serovars and a short period of their isolation were shown. In one hospital serovar K10 with some features of a "hospital" strain was isolated. The strains under study were sensitive to aminoglycosides, cephamesine, chlorhexidine, but resistant to semisynthetic penicillins and chloramine.     

<Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> antibiotic resistance identified by atomic force microscopy

In the last decade the detection of the resistance of bacteria to antibiotics treatment, developed by different kind of bacteria, is becoming a huge problem. We hereby present a different approach to the current problem of detection of bacteria resistance to antibiotics. Our aims were to use the atomic force microscopy (AFM) to investigate bacteria morphological changes in response to antibiotics treatment and explore the possibility of reducing the time required to obtain information on their resistance. In particular, we studied Klebsiella pneumoniae bacteria provided by the Lavagna Hospital ASL4 Liguria (Italy), where there are cases linked with antibiotics resistance of the Klebsiella pneumoniae. By comparing AFM images of bacteria strains treated with different antibiotics is possible to identify unambiguously the Klebsiella pneumoniae strains resistant to antibiotics. In fact, the analysis of the AFM images of the antibiotic-sensitive bacteria shows clearly the presence of morphological alterations in the cell wall. While in the case of the antibiotic-resistant bacteria morphological alterations are not present. This approach is based on an easy and potentially rapid AFM analysis.     

Antibiotic susceptibility, serum response and surface properties of Klebsiella species

Altogether 130 clinical isolates of five Klebsiella species (K. pneumoniae, K. planticola, K. oxytoca, K. ornithinolytica and K. terrigena) were characterized, for their susceptibility to five antibiotics, for susceptibility to serum bactericidal activity and for their hydrophobic properties. All strains exhibited ampicillin resistance. Ampicillin/sulbactam, gentamicin and ofloxacin showed effectiveness in 63.1, 67.7 and 71.5% of the Klebsiella isolates. K. planticola manifested the highest level of resistance to these antibiotics. The majority of Klebsiella strains (93.9%) were susceptible to cefuroxime. Sixty-four strains (49.2%) were serum resistant and intermediate serum sensitivity was shown by 57 strains (43.8%). A high percentage of serum resistant strains (65%) was found in K. planticola. Moderately hydrophobic properties determined by adherence of bacteria to xylene were demonstrated in 25 strains (19.2%).     

2011～2015年医院呼吸内科常见革兰阴性菌耐药性分析与临床用药探讨

目的：分析2011～2015年我院呼吸内科常见革兰阴性菌的分布情况及耐药性特点,为规范抗生素的合理使用提供依据。方法选用法国梅里埃公司生产的ID 32GD试剂盒,应用ATB Expression全自动分析系统对细菌进行鉴定与药敏试验。结果2011～2015年在我院呼吸内科常见的病原菌分别为肺炎克雷伯菌（31．0％）、大肠埃希菌（21．2％）、铜绿假单胞菌（23．2％）与鲍曼不动杆菌（14．8％）。肺炎克雷伯菌对碳青霉烯类的耐药率几乎为0,对单纯的头孢菌素耐药率相对较高,但对含β‐内酰胺酶抑制剂的头孢菌素类以及阿米卡星有较强的抗菌活性;大肠埃希菌对哌拉西林耐药率高,但对含酶青霉素类有较强的抗菌活性,对碳青霉烯类的耐药率连续5年来均为零;铜绿假单胞菌对磺胺类耐药性较高,对头孢三代、头孢四代及含酶青霉素类与含酶头孢类均有较强的抗菌活性,但敏感性呈逐年下降趋势;鲍曼不动杆菌的耐药率呈逐年上升趋势,仅对含酶的头孢哌酮／舒巴坦敏感（约80％）。结论医院呼吸内科革兰阴性菌仍以肺炎克雷伯菌、大肠埃希菌、铜绿假单胞菌、鲍曼不动杆菌为主。鲍曼不动杆菌耐药性逐年增长的问题已日趋严重,相关部门应定期监测细菌耐药趋势,指导临床合理用药,特别是合理应用抗生素以避免细菌耐药及减少细菌耐药性的发生。     

Antibiotic resistance and putative origin of Staphylococcus aureus and Klebsiella strains isolated from the children with intestinal dysbacteriosis

The results of the statistical treatment of data on the analyses of 766 children, the residents of Moscow, for dysbacteriosis are presented; of these children, 34 were aged up to 1 month and 732, from 1 month to 1 year. This study revealed that in the fist year of life in children with dysbacteriosis the dominating bacterial species were S. aureus, bacteria of the genus Klebsiella and fungi of the genus Candida. From the intestine of children aged up to 1 month S. aureus and Klebsiella were isolated more often than from children aged up to 1 year. The results of the study of antibioticograms demonstrated that 21.6% of S. aureus strains and 74.4% of Klebsiella strains were multiresistant to antibiotics. Taking into account the fact that multiresistance to antibiotics was characteristic of hospital strains, the suggestion was made that the isolated strains were of hospital origin and such strains could colonize the intestine of children in maternity hospitals.     

新生儿院内获得性肺炎患者病原体特征及 危险因素分析

目的分析院内新生儿获得性肺炎患者病原体分布、药敏情况和相关危险因素,并探讨其临床意义。方法收集2015年10月至2017年4月于我院接受治疗的100例院内获得性肺炎新生儿为观察组。选择同期于我院出生的未发生院内获得性肺炎的新生儿100例为对照组。采集观察组患儿下呼吸道分泌物,利用全自动微生物分析仪进行菌株种类和数量鉴定,利用K-B纸片扩散法进行药敏试验。对院内获得性肺炎的危险因素进行单因素回归分析。结果 100例院内获得性肺炎新生儿下呼吸道分泌物共检测出病原微生物168株,其中细菌135株(80.36%),病毒30株(17.86%),真菌3株(1.78%)。细菌中革兰阴性菌116株(69.05%),以大肠埃希菌(49株,29.17%)及肺炎克雷伯菌(42株,25.00%)为主;革兰阳性菌19株(11.31%),以金黄色葡萄球菌(10株,5.95%)为主。病毒以呼吸道合胞病毒(23株,13.69%)为主。大肠埃希菌及肺炎克雷伯菌均对常用抗生素具有极高的耐药性,包括氨苄西林、头孢哌酮、头孢曲松及头孢唑林等;对氨基糖苷类、喹诺酮类、碳青霉烯类抗生素高度敏感。新生儿的住院天数、分娩方式及吸痰均与院内获得性肺炎的发生有密切联系,差异均有统计学意义(P0.05)。结论新生儿院内获得性肺炎的致病菌以革兰阴性菌为主,临床治疗时可适当选用氨基糖苷类、喹诺酮类、碳青霉烯类抗生素。住院天数、剖腹产及吸痰均为新生儿院内获得性肺炎的危险因素,应加强对上述危险因素的保护。     

Catabolism of amino acids in bacteria of the genus Klebsiella

Propagation and activity level of 18 enzymes catalyzing deamination reactions of dicarboxylic and oxyamino acids and enzymes of amino acid reamination and amino acid N-acyl-derivatives' deacylation have been studied in Klebsiella bacteria. Klebsiella the most actively utilizes serin, threonine, aspartic and glutamic acids and aromatic amino acids. The first three amino acids are utilized by deamination, aromatic acids- in aminotransferase reaction with alpha-ketoglutaric acid, glutamic acid--by deamination and decarboxylation. Besides, Klebsiella actively deacylates N-acyl-derivatives of amino acids.     

工业腐败微生物种属特性及抗药性分析

为科学治理工业领域中微生物对杀菌剂的抗药性,从工业产品、原料及水样中采集腐败微生物,细菌按照《常见细菌系统鉴定手册》、API鉴定系统及16SrDNA序列分析,真菌按《真菌鉴定手册》及18SrDNA序列分析分别进行鉴定;通过测定杀菌剂的最小抑制浓度(MIC)来评估微生物抗药性水平。结果显示,腐败微生物中革兰氏阴性细菌约占46.91%,主要包括假单胞菌属、肠杆菌属、气单胞菌属、克雷伯氏菌属等;革兰氏阳性菌约占32.71%,主要种属为芽孢杆菌属、微杆菌属、李斯特氏菌属及球菌等;真菌约占12%,主要包括青霉属、木霉属和曲霉属。MIC测试结果显示,主要抗药性微生物为假单胞菌属,约占33.78%,平均抗性水平达到36mg/L,且传代不稳定。结论认为,工业上微生物污染主要由细菌耐药性引起,细胞膜结构及细菌生物膜的形成在该类杀菌剂抗药性产生的过程中起重要作用。     

Cyclitol utilization associated with the presence of Klebsielleae in botanical environments.

Bacteria of the tribe Klebsielleae are capable of metabolizing the cyclitols myoinositol, sequoyitol, and pinitol, which are present in aqueous extracts of redwood. Of the combined Klebsiella isolates from clinical and environmental origins, 100% (138/138), 97% (34/35), and 86% (119/138) fermented inositol, sequoyitol, and pinitol, respectively. These compounds were also used as a sole source of carbon and energy by Klebsiella. Similar results were obtained with Enterobacter isolates, but most other enteric bacteria tested could not metabolize cyclitols. Strains of Klebsiella multiplied to levels exceeding 10(5)/ml in aqueous extracts of nonsterile redwood within 6 days. Most other enteric bacteria did not grow in these extracts. Cyclitol metabolism was found to correlate well with the ability to multiply in redwood extract in the presence of cyclitol-negative indigenous bacteria. The capacity to use cyclitols, which are present in a variety of plant material, might afford Klebsielleae of both environmental and clinical origins an advantage in competing for nutrients and colonizing botanical environments.     

Cyclitol utilization associated with the presence of Klebsielleae in botanical environments.

Bacteria of the tribe Klebsielleae are capable of metabolizing the cyclitols myoinositol, sequoyitol, and pinitol, which are present in aqueous extracts of redwood. Of the combined Klebsiella isolates from clinical and environmental origins, 100% (138/138), 97% (34/35), and 86% (119/138) fermented inositol, sequoyitol, and pinitol, respectively. These compounds were also used as a sole source of carbon and energy by Klebsiella. Similar results were obtained with Enterobacter isolates, but most other enteric bacteria tested could not metabolize cyclitols. Strains of Klebsiella multiplied to levels exceeding 10(5)/ml in aqueous extracts of nonsterile redwood within 6 days. Most other enteric bacteria did not grow in these extracts. Cyclitol metabolism was found to correlate well with the ability to multiply in redwood extract in the presence of cyclitol-negative indigenous bacteria. The capacity to use cyclitols, which are present in a variety of plant material, might afford Klebsielleae of both environmental and clinical origins an advantage in competing for nutrients and colonizing botanical environments.     

呼吸道感染患者多重耐药菌肺炎克雷伯菌的耐药及危险因素分析

摘要 目的：探讨与分析呼吸道感染患者多重耐药菌肺炎克雷伯菌的耐药及危险因素。方法：选择2015年1月到2020年2月本院诊治的呼吸道感染患者65例作为研究对象,收集患者的临床样本进行细菌分离与耐药分析,调查患者的临床资料并进行危险因素分析。结果：在呼吸道感染患者65例中,分离出多重耐药菌肺炎克雷伯菌32株,占比49.2 %,其中下呼吸道、上呼吸道、灌洗液、血液标本分别占50.0 %、9.4 %、25.0 %、6.3 %。32株多重耐药菌肺炎克雷伯菌对头孢曲松、头孢呋辛、氨苄西林、头孢吡肟、头孢噻肟的耐药率分别为71.9 %、87.5 %、96.9 %、84.4 %、81.3 %,对阿米卡星、头孢替坦、左氧氟沙星、亚胺培南、环丙沙星的敏感率分别为59.4 %、68.8 %、81.3 %、75.0 %、81.3 %。非条件 Logistic回归分析显示血型A型、碳青霉烯类抗菌药物使用、引流、机械通气、糖尿病等为导致多重耐药菌肺炎克雷伯菌感染的独立危险因素(P<0.05)。结论：多重耐药菌肺炎克雷伯菌感染在呼吸道感染患者中比较常见,对头孢呋辛、氨苄西林的耐药率比较高,对左氧氟沙星、环丙沙星的敏感率比较高,血型A型、碳青霉烯类抗菌药物使用、引流、机械通气、糖尿病等为导致多重耐药菌肺炎克雷伯菌感染的独立危险因素。     

Genetic diversity and plant growth promoting traits of diazotrophic bacteria isolated from two Pennisetum purpureum Schum. genotypes grown in the field

Background and aims

Some elephant grass (Pennisetum purpureum) genotypes are able to produce large amounts of biomass and accumulate N derived from BNF when growing in soil with low N levels. However, information about the diazotrophic bacteria colonizing this C4 plant is still very scarce. This study aimed to characterize the plant growth promoting traits of a fraction of culturable diazotrophs colonizing the genotypes CNPGL F06-3 and Cameroon.

Methods

A total of 204 isolates were obtained from surface sterilized leaves, stems and roots after culturing on five different N-free semisolid media. These were then analyzed by BOX-PCR, and the 16S rRNA and nifH sequences of representative isolates were obtained. The functional ability of the isolates to reduce acetylene, produce indole and to solubilize phosphate was also determined.

Results

The diazotrophic bacterial population varied from 10² up to 10⁶ bacteria g^?1 fresh tissues of both genotypes. The BOX-PCR analysis suggested a trend in the genetic diversity among the 204 diazotrophic strains colonizing the different genotypes and plant tissues. Sequencing of 16S rRNA fragments confirmed the presence of Azospirillum brasilense and Gluconacetobacter diazotrophicus and revealed for the first time the occurrence of G. liquefaciens, G. sacchari, Burkholderia silvatlantica, Klebsiella sp., Enterobacter cloacae and E. oryzae in elephant grass. Interestingly, several nifH sequences from isolates identified as G. liquefaciens and G. sacchari showed homologies with nifH sequences of Enterobacter species. The majority of the isolates (97%) produced indole compounds, 22% solubilized phosphate and 6.4% possessed both characteristics.

Conclusions

The results showed the occurrence of novel diazotrophic bacterial species colonizing different tissues of both genotypes of elephant grass. In addition, the study revealed the presence of several bacteria with growth promoting traits, and highlighted their potential to be exploited as biofertilizers.     

Major structural proteins of type 1 and type 3 Klebsiella fimbriae are effective protein carriers and immunogens in conjugates as revealed from their immunochemical characterization

Fimbriae are filamentous structures present on the cell surface of many bacteria, including genus Klebsiella. The use of fimbriae as protein carriers in conjugates may allow to formulate effective multivalent vaccines and suitable diagnostics. However, the evidences have been reported that fimbriae may enhance the inflammatory response. This prompted us to examine the degree of cytokine induction by the type 1 and type 3 Klebsiella fimbriae and their conjugates. Fimbriae were assessed as carrier proteins for Escherichia coli K12 endotoxin core oligosaccharide. MALDI-MS revealed the molecular mass of fimbrial monomer major protein, which was 15,847 Da for type 1 and 18,574 Da for type 3 fimbriae of Klebsiella. These two types of fimbriae were moderate inductors of IL-6 and interferon and almost inactive with regard to the stimulation of TNF when tested in human whole blood assay. Coupling of fimbriae with E. coli K12 core oligosaccharide gave immunogenic conjugates with respect to a saccharide ligand and protein carrier, although only 10% of the pilin monomers possessed the attached oligosaccharide. Rabbit antiserum reacted with a broad spectrum of lipopolysaccharides, as measured by ELISA and immunoblotting assays. The antibodies against glycoconjugates were bactericidal for the wild, S-type bacteria of some species. Regarding the induction of cytokines by conjugates only the TNF level was noticeably elevated. These results prompt for the practical use of fimbriae, as effective protein carriers for conjugates to obtain broad-spectrum antisera for diagnostic applications.     

A specific property of bacteria in the genus Klebsiella--a color reaction with 5-aminosalicylic acid in the nutrient medium

For the first time bacteria of the genus Klebsiella have been found to possess a specific property, characteristic of this genus only, i.e. the capacity of giving color reaction with 5-aminosalicylic acid. This reaction can be observed in all Klebsiella species under study: K. pneumoniae (94.4 +/- 2.3%), K. ozaenae (93.3 +/- 4.5%), K. rhinoscleromatis (100%), K. oxytoca (88.0 +/- 4.9%), K. mobilis (92-5 +/- 4.3% of the strains). In all other bacteria under study (40 species, 22 genera and 7 families) the reaction is negative. The test for the color reaction with 5-aminosalicylic acid confirms the belonging of K. mobilis (Enterobacter aerogenes) to the genus Klebsiella, thus making it possible to simplify and accelerate the identification of Klebsiella.     

Hyperphagocytic haemocytes in Manduca sexta

We have discovered a new type of haemocyte in the larval stage of the tobacco hornworm moth Manduca sexta that has extreme phagocytic ability; each cell can engulf up to 500 bacteria. This level of phagocytosis may be unprecedented among animal cells. Although these hyperphagocytic cells (HP) only represent about 1% of the circulating haemocytes, they are responsible for sequestering the majority of the bacteria by circulating haemocytes when non-pathogenic, heat-killed Escherichia coli are injected into the haemolymph. Extreme phagocytosis by HP is not limited to Gram-negative bacteria since heat-killed Staphylococcus aureus as well as positively and negatively charged microspheres are also highly phagocytosed. Evidence is presented to show that phagocytosis by HP is involved in the early stages of nodule formation in infected insects. In addition, HP are also present in non-infected insects, characterised by their distinctive spreading morphology, which becomes impaired following hyperphagocytosis of bacteria. This is the first time that a dedicated "professional" phagocytic class of haemocyte has been reported for an invertebrate. The importance of these specialised cell types in the M. sexta immune response and their role in nodule formation is discussed.     

Diversity and vertical distribution of magnetotactic bacteria along chemical gradients in freshwater microcosms

The vertical distribution of magnetotactic bacteria along various physico-chemical gradients in freshwater microcosms was analyzed by a combined approach of viable cell counts, 16S rRNA gene analysis, microsensor profiling and biogeochemical methods. The occurrence of magnetotactic bacteria was restricted to a narrow sediment layer overlapping or closely below the maximum oxygen and nitrate penetration depth. Different species showed different preferences within vertical gradients, but the largest proportion (63-98%) of magnetotactic bacteria was detected within the suboxic zone. In one microcosm the community of magnetotactic bacteria was dominated by one species of a coccoid "Alphaproteobacterium", as detected by denaturing gradient gel electrophoresis in sediment horizons from 1 to 10 mm depth. Maximum numbers of magnetotactic bacteria were up to 1.5 x 10(7) cells/cm3, which corresponded to 1% of the total cell number in the upper sediment layer. The occurrence of magnetotactic bacteria coincided with the availability of significant amounts (6-60 microM) of soluble Fe(II), and in one sample with hydrogen sulfide (up to 40 microM). Although various trends were clearly observed, a strict correlation between the distribution of magnetotactic bacteria and individual geochemical parameters was absent. This is discussed in terms of metabolic adaptation of various strains of magnetotactic bacteria to stratified sediments and diversity of the magnetotactic bacterial communities.     

Intestinal enterobacteria of the hibernating Apis mellifera mellifera L. bees

Dynamics of enterobacteria of normal intestinal microflora was studied in Apis mellifera mellifera L. bees hibernating under snow in the Western Urals. The cell numbers (N) of the predominant species Klebsiella oxytoca increased from 10-10(6) CFU/bee in November 2004 to 10(4)-10(7) CFU/bee in March 2005; its frequency of occurrence (P) increased from 92 to 100%. Increase of Providencia rettgeri (11.2004: N up to 10(6), P 25%; 03.2005: N 10(2)-10(6), P 80%) was accompanied by the substitution of Morganella morganii (11.2004: N up to 10(6), P 25%) with Proteus vulgaris (03.2005: N up to 10(5), P 8%). By spring, Hafnia alvei and Citrobacter sp., which are pathogenic to bees, disappeared (11.2004: N up to 10(5), P 13 and 10%, respectively). Endophytic species Pantoea agglomerans, Leclecria sp., and other representatives of the "Enterobacter agglomerans" group were present in November and after the first emergence in spring (N up to 10(5); November: P 15%; April: P 23%). In April, the number of enterobacteria decreased to 10(5), and P. rettgeri became the predominant species (P 54%) instead of K. oxytoca (P 43%).     

Factors regulating the density of bacteria in process waters of a paper mill

Variations in the numbers of total colonies of Klebsiella pneumoniae, Acinetobacter spp. and pseudomonads were investigated in process waters of a paper mill in southern Finland. Variations were related to independent parameters, namely temperature, pH, redox potential and production of offset paper, by using multiple regression analysis. Temperature was the most significant regressor variable and was negatively correlated with bacterial counts. It accounted for up to about 80% of the variance in bacterial counts in various parts of the process. The significance of temperature was due to its fluctuations in a critical range, above and below the maxima for bacterial growth. The pH level was also significant for total colony count and for K. pneumoniae. Redox potential and the production of offset paper were of significance for Acinetobacter spp. Washing the paper machine with water and lye decreased the numbers of bacteria in process waters.     

Fimbrial hemagglutinins in Klebsiella pneumoniae

Eleven Klebsiella pneumoniae strains were isolated from urine specimens which were examined for their ability to produce hemagglutinins (HAs). Bacteria were grown under various culture conditions. Suspension of bacteria grown in broth or on Phosphate-buffered nutrient agar (PBA) were tested for agglutination in the presence and absence of 2% (w/v) D-mannose, on rockedtiles at 4 degrees C and ambient temperature with mangroup-O(M), fowl(F), ox(O), guinea-pig(G), horse (H), rabbit (R) and sheep (S) erythrocytes and tannic acid treated, but not fresh oxen erythrocytes. Each of the 11 strains was hemagglutinating. Ten strains (99%) producing two or three hemagglutinins (HAs), were multiple hemagglutinating. One strain produced only mannose-resistant, Klebsiella, the "Tanned ox hemagglutinin" (MR/K-HA). Solely mannose-sensitive hemagglutinin (MS/HA) was not produced by any of the strains. No mannose-sensitive hemagglutinating strains acted on sheep erythrocytes. Three main kinds of hemagglutinin (HA) were detected. These were: (a) a mannose-sensitive hemagglutinin (MS-HA); (b) a mannose resistant, Klebsiella, the "tanned ox hemagglutinin (MR/K-HA); (c) mannose-resistant, Proteus hemagglutinin (MR/P-HA). All strains (100%) produced MR/K-HA, 45% of MR/K-HA+ strains produced MR/P-HA at 37 degrees C and 99% of all strains produced MS/HA, MR/P-HA activity was never dependent on MR/K-HA: Electronmicroscopic examination of bacteria showed that all strains were fimbriate.     

Profiling of the bacteria responsible for pyogenic liver abscess by 16S rRNA gene pyrosequencing

Pyogenic liver abscess (PLA) is a severe disease with considerable mortality and is often polymicrobial. Understanding the pathogens that cause PLA is the basis for PLA treatment. Here, we profiled the bacterial composition in PLA fluid by pyrosequencing the 16S ribosomal RNA (rRNA) gene based on next-generation sequencing (NGS) technology to identify etiological agents of PLA and to provide information of their 16S rRNA sequences for application to DNA-based techniques in the hospital. Twenty patients with PLA who underwent percutaneous catheter drainage, abscess culture, and blood culture for isolates were included. Genomic DNAs from abscess fluids were subjected to polymerase chain reaction and pyrosequencing of the 16S rRNA gene with a 454 GS Junior System. The abscess and blood cultures were positive in nine (45%) and four (20%) patients, respectively. Pyrosequencing of 16S rRNA gene showed that 90% of the PLA fluid samples contained single or multiple genera of known bacteria such as Klebsiella, Fusobacterium, Streptococcus, Bacteroides, Prevotella, Peptostreptococcus, unassigned Enterobacteriaceae, and Dialister. Klebsiella was predominantly found in the PLA fluid samples. All samples that carried unassigned bacteria had 26.8% reads on average. We demonstrated that the occurrence of PLA was associated with eight known bacterial genera as well as unassigned bacteria and that 16S rRNA gene sequencing was more useful than conventional culture methods for accurate identification of bacterial pathogens from PLA.