Molecular epidemiology of ESbetaL producing P. mirabilis strains from a long-term care and rehabilitation facility in Italy

We report the detection of multidrug resistant ESbetaL producing Proteus mirabilis isolates from a long-term care and rehabilitation facility (LTCRF) in Northern Italy. 53% of the collected P. mirabilis strains were ESbetaL producers. PCR and sequencing techniques confirmed the presence of the bla(TEM-92) and bla(CMY-16) resistance genes in 23/26 (88.5%) and 3/26 (11.5%) of the ESbetaL producers respectively. PFGE showed that the TEM-92 beta-lactamase producing isolates were not clonally related, indicating the presence of at least four different clonal lineages (A, B, C, D), whereas all the CMY-16 enzyme producers belonged in the same lineage. The bla(TEM-92) and bla(CYY-16) determinants were distributed in seven different wards, but in three of them they coexisted. Our results show that the most patients are co-colonized by ESbetaLs producing P. mirabilis strains at the time of admission to an LTCRF. An effective strategy to curtail the spread of ESbetaLs mediated resistance in LTCRFs could be to activate sourveillance programs to monitor routinely the entry of resistant bacteria.     

Species-specific detection of Proteus vulgaris and Proteus mirabilis by the polymerase chain reaction

Sets of primers for the species-specific detection of P. mirabilis and P. vulgaris by the polymerase chain reaction (PCR) were developed. As targets for these primers beta-lactamase and 16S rRNA gene fragments were chosen on the basis of the multiple leveling of the sequences of the DNA of all known P. mirabilis and P. vulgaris isolates. For differential detection oligonucleotides were selected in such a way that primers, specific for P. vulgaris, contained the non-paired nucleotide for P. mirabilis isolate at the 3'-end, and all other nucleotides were complementary to the beta-lactamase gene fragment. Primers, specific for gene 16S rRNA of P. mirabilis, contained the non-paired nucleotide for P. vulgaris isolates at the 3'-end. Standard PCR was carried out for 6 P. mirabilis and P. vulgaris strains. The use of PCR species-specific primers to P. vulgaris DNA made it possible to amplify the DNA fragment of the expected length only for P. vulgaris isolates, while the result of PCR for P. mirabilis was negative. PCR with primers specific to P. mirabilis permitted the detection of amplicon sized 101 nucleotides pairs only for P. mirabilis strains. These primers were optimized so as to use them in the specific differentiation of closely related P. mirabilis and P. vulgaris species by multiplex PCR. Genus-specific primers permitted the detection of bacterial gyrB gene of the genus Proteus were developed also.     

Occurrence of 16s rRNA methylase ArmA producing Enterobacteriaceae in a general hospital in Warsaw, Poland

Resistance to gentamicin, amikacin and kanamycin was screened in 270 clinical isolates of Enterobacteriaceae originated from April 19 to May 19, 2010 in a regular hospital in Warsaw, Poland. Most of the isolated bacteria were considered pathogenic. Nineteen isolates (7%) were simultaneously resistant to two or three of the tested aminoglycosides. MICs of the three aminoglycosides ranged form 128 to 1024 mcg/ml for six isolates. These isolates were suspected to produce 16S rRNA methylase. Genes encoding for three methylases reported in Europe: ArmA, RmtB and RmtC were searched by PCR. The armA gene was detected in all of the six isolates. This group encompassed Enterobacter cloacae (n=4), Klebsiella pneumoniae (n=1) and Proteus mirabilis (n=1). Five isolates of this group carried the bla(CAX-M) gene for CTX-M type ESBL. The remaining isolate E. cloacae DM0340 was ESBL negative and lacked bla(CRX-M) that may suggest an altered genetic environment of the armA gene in this isolate. Our results showed that 2.2% of the tested isolates produced 16S rRNA methylase ArmA. This finding may argue for a high incidence of ArmA producing Enterobacteriaceae in Poland when compared to reports from other European countries.     

头孢西丁耐药肺炎克雷伯菌AmpC基因和ESBLs检测及耐药性分析

目的了解深圳地区头孢西丁耐药肺炎克雷伯菌头孢菌素酶(AmpC酶)基因型分布、产超广谱β-内酰胺酶(ESBLs)的情况及其耐药特点。方法收集深圳地区三家大型综合医院临床标本分离对头孢西丁耐药的肺炎克雷伯菌73株。用碱裂解法提取菌株的质粒,采用多重PCR扩增AmpC基因,应用DNA测序确定其基因型。并对所有菌株进行ESBLs表型确证试验;用K-B法对其进行药物敏感试验。结果 48株(65.8%)AmpC基因扩增阳性,经DNA测序显示,其中46株为DHA-1型,1株为CMY-2型,1株同时产DHA-1和CMY-2型;73株肺炎克雷伯菌中49株ESBLs阳性,其中36株AmpC基因和ESBLs均为阳性。AmpC和(或)ESBLs阳性菌株对多数药物的耐药率高于AmpC和ESBLs均阴性者。结论本地区头孢西丁耐药肺炎克雷伯菌质粒AmpC酶检出率高,基因型主要为DHA-1,同时产AmpC酶和ESBLs菌株较常见。     

奇异变形杆菌耐药性及亚胺培南不敏感株感染的临床特点研究

目的研究奇异变形杆菌的临床分布和耐药情况、亚胺培南不敏感奇异变形杆菌感染的临床特点。方法分析浙江大学医学院附属第一医院2013年1月至2013年12月分离的非重复奇异变形杆菌的药物敏感性、临床分布,回顾性分析亚胺培南不敏感奇异变形杆菌感染患者的临床资料、治疗及预后情况。结果2013年该院共分离107株奇异变形杆菌,以分离自尿液最多,其次为痰液;来源最多的是外科病房和重症监护病房。体外药敏显示：奇异变形杆菌对美罗培南、厄他培南、头孢吡肟、氨曲南、哌拉西林/他唑巴坦、头孢他啶、头孢哌酮/舒巴坦、阿米卡星等抗菌药物敏感性良好,敏感率达85%以上;对亚胺培南敏感率为80.4%;对头孢呋辛、环丙沙星、氨苄西林、头孢曲松、庆大霉素耐药率较高,超过30%;对呋喃妥因耐药率为99%。其中21株亚胺培南不敏感奇异变形杆菌对包括美罗培南、厄他培南在内的其他各类抗菌药物耐药率与亚胺培南敏感株基本相仿。亚胺培南不敏感奇异变形杆菌引起院内获得性感染主要发生在入住ICU、外科术后、广谱抗菌药物使用后、留置各类置管和梗阻性尿路疾病的患者,可引起泌尿系统、皮肤创面、腹腔、血流、生殖道等部位感染,表现为全身炎症反应及局部感染症状。选择敏感抗菌药物治疗后该部分患者预后良好。结论奇异变形杆菌对三、四代头孢菌素,β-内酰胺酶抑制剂合剂等抗生素敏感性良好。亚胺培南不敏感奇异变形杆菌对其他碳青酶烯类抗生素仍保持较高的敏感性。亚胺培南不敏感奇异变形杆菌所引起院内获得性感染主要发生在入住ICU、外科术后、广谱抗菌药物使用后、留置各类置管和梗阻性尿路疾病的患者,预后良好。     

Mechanisms of resistance to cephalosporin and emergence of O25b-ST131 clone harboring CTX-M-27 β-lactamase in extraintestinal pathogenic Escherichia coli from dogs and cats in Japan

Thirty-three cefazolin-resistant extraintestinal pathogenic Escherichia coli strains from companion animals were screened for bla(CMY-1) , bla(CMY-2) , bla(SHV) , bla(TEM) , and bla(CTX-M) genes. Extended-spectrum β-lactamase-producing strains were further characterized by O serotyping and multilocus sequence typing. It was found that 20 and 17 isolates harbored TEM-1 and CMY-2 β-lactamases, respectively, and 13 isolates harbored both β-lactamases. One isolate harbored DHA-1 β-lactamase. Eleven isolates were found to possess CTX-M β-lactamases (CTX-M-27 [n= 6], CTX-M-14 [n= 3], CTX-M-15 [n= 1], and CTX-M-55 [n= 1]). Of 11 CTX-M-positive strains, four strains were O25b-ST131 clones harboring CTX-M-27, and the remaining seven strains belonged to O6-ST127, ONT-ST354, O159-ST539, O1-ST648, O8-ST1642, O25b-ST2042, and ONT-ST2178.     

Giardia microti in pet Microtus guentheri: Evidence of a parasite never detected in Italy

The genus Giardia includes several species distinguished by morphological, biological and molecular features. Currently, eight species within the genus are retained as valid. In Italy no identification of Giardia species other than Giardia duodenalis has been so far reported. Fecal samples were collected from two Günther's Voles (Microtus guentheri) positive to Giardia cysts by microscopic investigation and immunofluorescence. The voles were born in Milan (Northern Italy) from two gravid females imported from the Netherlands and kept for sale in a pet shop in Varese (Northern Italy). Positive feces were subjected to a nested PCR to amplify a 18S rRNA fragment for molecular characterization. A phylogenetic analysis was conducted to compare the obtained sequence with those of all other Giardia species available in GenBank for the 18S locus, using the Maximum Likelihood (ML) method by R software. Sequence analyses unambiguously identified the isolates as belonging to G. microti, showing 99% of identity with those of its isolates available in GenBank. A well-defined cluster, supported by significant bootstrap values and corresponding to the G. microti cluster, including sequences obtained from M. guentheri, was evidenced in the ML tree, confirming species assignment. The present finding represents the first report of G. microti from pet animals in Italy.     

Some biological features of Proteus bacilli. 2. Haemolytic activities of Proteus mirabilis and Proteus vulgaris strains

The haemolytic activities of Proteus mirabilis and P. vulgaris strains were studied under different conditions. No filterable alpha haemolysin could be detected in P. mirabilis uropathogens provided from patients with urinary tract infections. Together with the results presented in the accompanying paper, in which three clinical isolates with temporary ability to produce a soluble haemolysin were described, the occurrence of alpha haemolytic P. mirabilis isolates did not exceed 3%. Cell bound beta haemolysin is present in nearly 35% of P. mirabilis urinary strains. Another kind of haemolytic activity was observed when P. mirabilis and P. vulgaris strains were grown in liquid media supplemented with erythrocytes. During the logarithmic growth phase nearly 100% of P. mirabilis and P. vulgaris strains of various origin haemolyzed 100-50% of erythrocytes. Except for Serratia, the other representatives of Enterobacteriaceae did not demonstrate such activity in the same conditions. The preliminary characteristics of this phenomenon is given.     

Bacteria recovered without subculture from infected human urines expressed iron-regulated outer membrane proteins

Abstract Twelve enteric bacterial strains were recovered by differential centrifugation of urines which were collected from clinically diagnosed and microbiologically confirmed cases of urinary tract infection. The outer membrane protein (OMP) profiles of the clinical isolates were then analysed by sodiumdodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found that 5 of the 12 isolates (3 Escherichia coli strains, 1 Klebsiella pneumoniae and 1 Proteus mirabilis strain) expressed 2 or more high M _r proteins in the range of 66000 to 85000. These high M _r proteins were expressed by the same organisms during growth in vitro in iron-restricted conditions but not in iron-sufficient media.
In addition, it was found that the major outer membrane proteins expressed by the clinical isolates varied considerably and that, in many cases, fresh isolates expressed fewer porin proteins than the same bacterial strains after growth in vitro in trypticase soy broth. This is thus the first evidence the E. coli, K. pneumoniae and P. mirabilis grow under iron-restricted conditions in the urinary tract of humans and that the outer membrane protein profile of clinical isolates differ from in vitro grown bacteria.     

Rapid Molecular Characterization of Acinetobacter baumannii Clones with rep-PCR and Evaluation of Carbapenemase Genes by New Multiplex PCR in Hospital District of Helsinki and Uusimaa

Multidrug-resistant Acinetobacter baumannii (MDRAB) is an increasing problem worldwide. Prevalence of carbapenem resistance in Acinetobacter spp. due to acquired carbapenemase genes is not known in Finland. The purpose of this study was to examine prevalence and clonal spread of multiresistant A. baumannii group species, and their carbapenemase genes. A total of 55 Acinetobacter isolates were evaluated with repetitive PCR (DiversiLab) to analyse clonality of isolates, in conjunction with antimicrobial susceptibility profile for ampicillin/sulbactam, colistin, imipenem, meropenem, rifampicin and tigecycline. In addition, a new real-time PCR assay, detecting most clinically important carbapenemase genes just in two multiplex reactions, was developed. The assay detects genes for KPC, VIM, IMP, GES-1/-10, OXA-48, NDM, GIM-1, SPM-1, IMI/NMC-A, SME, CMY-10, SFC-1, SIM-1, OXA-23-like, OXA-24/40-like, OXA-58 and ISAbaI-OXA-51-like junction, and allows confident detection of isolates harbouring acquired carbapenemase genes. There was a time-dependent, clonal spread of multiresistant A. baumannii strongly correlating with carbapenamase gene profile, at least in this geographically restricted study material. The new carbapenemase screening assay was able to detect all the genes correctly suggesting it might be suitable for epidemiologic screening purposes in clinical laboratories.     

Plasmid and Host Strain Characteristics of Escherichia coli Resistant to Extended-Spectrum Cephalosporins in the Norwegian Broiler Production

Escherichia coli resistant to extended-spectrum cephalosporins have been detected in the Norwegian broiler production, despite the fact that antimicrobial agents are rarely used. The genetic mechanism responsible for cephalosporin resistance is mainly attributed to the presence of the bla_CMY-2 gene encoding a plasmid-mediated AmpC-beta-lactamase (pAmpC). The aim of this study was to characterize and compare bla_CMY-2 containing Escherichia coli isolated from the intestinal flora of broilers and retail chicken meat (fillets) to identify possible successful clones and/or resistance plasmids widespread in the Norwegian broiler production. Methods used included PCR based phylotyping, conjugation experiments, plasmid replicon typing, pulsed-field gel electrophoresis, multiple locus variable-number tandem-repeats analysis and whole genome sequencing. The nucleotide sequence of an IncK plasmid carrying bla_CMY-2 was determined. Intestinal isolates displayed a higher degree of genetic diversity than meat isolates. A cluster of genetically related isolates belonging to ST38, phylogroup D, carrying bla_CMY-2 containing IncK plasmids was identified. Furthermore, genes encoding plasmid stability systems (relBE/stbDE and pndAC) were identified on the IncK plasmid. Single nucleotide polymorphism (SNP) analysis of a subset of isolates confirmed a close genetic relationship within the two most prevalent STs. The IncK plasmids within these two STs also shared a high degree of similarity. Cephalosporin-resistant E. coli with the same genetic characteristics have been identified in the broiler production in other European countries, and the IncK plasmid characterized in this study showed close homology to a plasmid isolated from retail chicken meat in the Netherlands. The results indicate that both clonal expansion and horizontal transfer of bla_CMY-2 containing plasmids contribute to dissemination of cephalosporin resistant E. coli in the broiler production. The presence of plasmid stability systems may explain why the IncK plasmid containing bla_CMY-2 is maintained and disseminated in the Norwegian broiler production in absence of selection pressure from the use of antimicrobial agents.     

IncA/C Plasmid-Mediated Spread of CMY-2 in Multidrug-Resistant Escherichia coli from Food Animals in China

Objectives

To obtain a broad molecular epidemiological characterization of plasmid-mediated AmpC β-lactamase CMY-2 in Escherichia coli isolates from food animals in China.

Methods

A total of 1083 E. coli isolates from feces, viscera, blood, drinking water, and sub-surface soil were examined for the presence of CMY-2 β-lactamases. CMY-2-producing isolates were characterized as follows: the bla _CMY-2 genotype was determined using PCR and sequencing, characterization of the bla _CMY-2 genetic environment, plasmid sizing using S1 nuclease pulsed-field gel electrophoresis (PFGE), PCR-based replicon typing, phylogenetic grouping, XbaI-PFGE, and multi-locus sequence typing (MLST).

Results

All 31 CMY-2 producers were only detected in feces, and presented with multidrug resistant phenotypes. All CMY-2 strains also co-harbored genes conferring resistance to other antimicrobials, including extended spectrum β-lactamases genes (bla _CTX-M-14 or bla _CTX-M-55), plasmid-mediated quinolone resistance determinants (qnr, oqxA, and aac-(6′)-Ib-cr), floR and rmtB. The co-transferring of bla _CMY-2 with qnrS1 and floR (alone and together) was mainly driven by the Inc A/C type plasmid, with sizes of 160 or 200 kb. Gene cassette arrays inserted in the class 1 or class 2 integron were amplified among 12 CMY-2 producers. CMY-2 producers belonged to avirulent groups B1 (n = 12) and A (n = 11), and virulent group D (n = 8). There was a good correlation between phylogenetic groups and sequence types (ST). Twenty-four STs were identified, of which the ST complexes (STC) 101/B1 (n = 6), STC10/A (n = 5), and STC155/B1 (n = 3) were dominant.

Conclusions

CMY-2 is the dominant AmpC β-lactamase in food animals and is associated with a transferable replicon IncA/C plasmid in the STC101, STC10, and STC155 strains.     

Occurrence of Pseudomonas syringae pv. Tomato race 1 in Italy on Pto Gene-Bearing Tomato Plants

Bacterial speck symptoms on leaves of the tomato cultivar Erminia Fl (Petoseed), heterozygous for the Pto resistant gene, were observed in June 1995 in Northern Italy. Using individual-lesion isolation, 8 bacterial isolates were obtained from the affected leaves, which were identified as Pseudomonas syringae pv. tomato by biochemical, physiological, nutritional and pathogenicity tests. When the differential cultivar Ontario 7710, homozygous for the Pto gene, was inoculated with the bacterial isolates, 4 of them provoked the typical bacterial speck symptoms and therefore belonged to race 1. This is the first occurrence of P. syringae pv. tomato race 1 in Italy and the first time this race has been isolated on a Pto gene-bearing tomato cultivar grown in open field.     

Identification of enzyme-producing thermophilic bacilli isolated from marine vents of Aeolian Islands (Italy)

Enzyme-producing thermophilic bacilli were isolated from different thermal sites located in and around Aeolian Islands (Italy) and characterised by both molecular and culture-based methods. Spore-forming bacteria with optimal growth from 45 to 70 °C were isolated from submarine vents and a geothermal well of Aeolian Islands (Italy). Eighteen selected strains were screened for extracellular enzyme presence by using nine substrates: Tween 20, Tween 80, tributyrin, soluble starch, xylan, dextran, carragenan, gelatine and casein. Sixteen isolates were able to grow at pH 9. The isolates were differentiated on the basis of restriction pattern of their amplified 16S rDNA (ARDRA) prior to 16S rRNA gene sequence analysis. On the basis of the most complete sequencing results strain V3 was identified asGeobacillus thermodenitrificans, most of isolates (10/14) was similar at high level (≥95%) to different reference strains of the speciesBacillus licheniformis. The remaining isolates, exhibiting sequence similarity below 95%, may represent novel species of the genusBacillus.     

A shift from acute to chronic spontaneous pyelonephritis in male MM mice associated with a change in the causal micro-organisms

Proteus mirabilis was the predominant cause of acute diabetes-associated pyelonephritis occurring spontaneously in male MM mice until they were segregated in a new environment. Thereafter Pasteurella pneumotropica and Streptococcus faecalis emerged collectively as the most common causal organisms, the pyelonephritis became more chronic and Proteus mirabilis isolates from faeces and urine produced atypical non-swarming colonies on blood agar plates. This did not account for the reduced pathogenicity of Proteus mirabilis; when MM males were returned to the original environment the pyelonephritis again became acute but was associated with the atypical type of Proteus mirabilis although the normal type was abundant in the environment. The MM mice were Caesarean-derived and cross-fostered shortly before their transfer to the second environment, which probably accounts for their changed microbial status, but the reason for the emergence of the atypical type of Proteus mirabilis is not understood. The acute nature of the male MM pyelonephritis when caused by Proteus mirabilis parallels the situation described in other animals and humans.     

Structural basis for the extended substrate spectrum of CMY-10, a plasmid-encoded class C beta-lactamase

The emergence and dissemination of extended-spectrum (ES) beta-lactamases induce therapeutic failure and a lack of eradication of clinical isolates even by third-generation beta-lactam antibiotics like ceftazidime. CMY-10 is a plasmid-encoded class C beta-lactamase with a wide spectrum of substrates. Unlike the well-studied class C ES beta-lactamase from Enterobacter cloacae GC1, the Omega-loop does not affect the active site conformation and the catalytic activity of CMY-10. Instead, a three-amino-acid deletion in the R2-loop appears to be responsible for the ES activity of CMY-10. According to the crystal structure solved at 1.55 A resolution, the deletion significantly widens the R2 active site, which accommodates the R2 side-chains of beta-lactam antibiotics. This observation led us to demonstrate the hydrolysing activity of CMY-10 towards imipenem with a long R2 substituent. The forced mutational analyses of P99 beta-lactamase reveal that the introduction of deletion mutations into the R2-loop is able to extend the substrate spectrum of class C non-ES beta-lactamases, which is compatible with the isolation of natural class C ES enzymes harbouring deletion mutations in the R2-loop. Consequently, the opening of the R2 active site by the deletion of some residues in the R2-loop can be considered as an operative molecular strategy of class C beta-lactamases to extend their substrate spectrum.     

Bacteriophage Typing of Proteus mirabilis, Proteus vulgaris, and Proteus morganii

A bacteriphage typing scheme for differentiating Proteus isolated from clinical specimens was developed. Twenty-one distinct patterns of lysis were seen when 15 bacteriophages isolated on 8 Proteus mirabilis, 1 P. vulgaris, and 1 P. morganii were used to type 162 of 189 (85.7%) P. mirabilis and P. vulgaris isolates. Seven phages isolated on 3 P. morganii were used to type 13 of 19 (68.4%) P. morganii isolates. Overall, 84.1% of the 208 isolates were lysed by at least 1 phage at routine test dilution (RTD) or 1,000 x RTD. Fifty isolates, retyped several weeks after the initial testing, showed no changes in lytic patterns. The phages retained their titers after storage at 4 C for several months. A computer analysis of the data showed that there was no relationship between the source of the isolate and bacteriophage type. This bacteriophage typing system may provide epidemiological information on strains involved in human infections.     

Aggregative adherence of uropathogenic Proteus mirabilis to cultured epithelial cells

Proteus mirabilis is an important cause of urinary tract infection (UTI) in patients with complicated urinary tracts. Thirty-five strains of P. mirabilis isolated from UTI were examined for the adherence capacity to epithelial cells. All isolates displayed the aggregative adherence (AA) to HEp-2 cells, a phenotype similarly presented in LLC-MK(2) cells. Biofilm formation on polystyrene was also observed in all strains. The mannose-resistant Proteus-like fimbriae (MR/P), Type I fimbriae and AAF/I, II and III fimbriae of enteroaggregative Escherichia coli were searched by the presence of their respective adhesin-encoding genes. Only the MR/P fimbrial subunits encoding genes mrpA and mrpH were detected in all isolates, as well as MR/P expression. A mutation in mrpA demonstrated that MR/P is involved in aggregative adherence to HEp-2 cells, as well as in biofilm formation. However, these phenotypes are multifactorial, because the mrpA mutation reduced but did not abolish both phenotypes. The present results reinforce the importance of MR/P as a virulence factor in P. mirabilis due to its association with AA and biofilm formation, which is an important step for the establishment of UTI in catheterized patients.     

High levels of genetic variation exist in Aspergillus niger populations infecting Welwitschia mirabilis hook

Aspergillus niger is an asexual, haploid fungus which infects the seeds of Namibia's national plant, Welwitschia mirabilis, severely affecting plant viability. We used 31 randomly amplified polymorphic DNA markers to assess genetic variation among 89 A. niger isolates collected from three W. mirabilis populations in the Namib Desert. While all isolates belonged to the same vegetative compatibility group, 84% were unique genotypes, and estimates of genotypic evenness and Simpson's index of diversity approached 1.0 in the three populations. Analysis of molecular variance revealed that 78% of the total variation sampled was among isolates from individual W. mirabilis plants. Lower, but significant, amounts of variation detected among isolates from different plants (12%) and different sites (10%) also indicated some site- and plant-level genetic differentiation. Total gene diversity (H(T) = 0.264) was mostly attributable to diversity within populations (H(S) = 0.217); the relatively low level of genetic differentiation among the sites (G(ST) = 0.141) suggests that gene flow is occurring among the three distant sites. Although sexual reproduction has never been observed in this fungus, parasexuality is a well-known phenomenon in laboratory strains. We thus attribute the high levels of genetic variation to parasexuality and/or wind-facilitated gene flow from an as of yet undocumented broader host range of the fungus on other desert vegetation. Given the apparent ease of transmission, high levels of genetic diversity, and potentially broad host range, A. niger infections of W. mirabilis may be extremely difficult to control or prevent.