Enterobacterial Repetitive Intergenic Consensus Primers Research Articles

American Foulbrood in the Czech Republic: ERIC II Genotype of Paenibacillus Larvae Is Prevalent.

American foulbrood (AFB) is a dangerous disease of honeybees (Apis mellifera) caused by the spore-forming bacterium Paenibacillus larvae. According to the ERIC (enterobacterial repetitive intergenic consensus) classification, five genotypes are distinguished, i.e., I, II, III, IV, and V, which differ in their virulence and prevalence in colonies. In the Czech Republic, AFB prevalence is monitored by the State Veterinary Administration; however, the occurrence of specific P. larvae genotypes within the country remains unknown. In this study, our aim was to genotype field P. larvae strains collected in the Czech Republic according to the ERIC classification. In total, 102 field isolates from colonies with AFB clinical symptoms were collected from various locations in the Czech Republic, and the PCR genotypization was performed using ERIC primers. We confirmed the presence of both ERIC I and II genotypes, while ERIC III, IV, and V were not detected. The majority of samples (n = 82, 80.4%) were identified as ERIC II, while the ERIC I genotype was confirmed only in 20 samples (19.6%). In contrast to other European countries, the ERIC II genotype is predominant in Czech honeybee colonies. The ERIC I genotype was mostly detected in border regions close to Poland, Slovakia, and Austria.

Virulence and DNA fingerprinting analysis of Xanthomonas oryzae pv. oryzae identify a new pathotype in Guangxi, South China.

Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive diseases affecting rice worldwide. However, little is known about the population structure of this organism in Guangxi Zhuang Autonomous Region, South China. Here, pathotypic and DNA fingerprint analyses were conducted to characterize the isolates of Xoo collected from rice leaves in five districts of the region from 2013 to 2016. Their pathogenicity was tested by leaf clipping, and the DNA fingerprints were analyzed by repetitive sequence-based polymerase chain reaction and endogenous insertion sequence element-based polymerase chain reaction assays using the repetitive extragenic palindromic sequence and enterobacterial repetitive intergenic consensus primers, respectively. Pathogenicity assays of 70 representative isolates were conducted using a series of near-isogenic lines and two new pathotypes were identified. All the pathotypes were found to be incompatible with xa5 and Xa7. One pathotype was virulent to Xa14, Xa21, and Xa23, whereas another virulent to Xa21 and Xa23, but incompatible with Xa14. A dendrogram generated for the data sets obtained from DNA fingerprinting suggested the prevalence of high genetic diversity of Xoo throughout Guangxi, and no association between the molecular haplotypes and pathotypes was identified.

Molecular Characterization of Vibrio cholerae Isolated From Clinical Samples in Kurdistan Province, Iran.

Background:Vibrio cholerae causes diarrhoeal disease that afflicts thousands of people annually. V. cholerae is classified on the basis of somatic antigens into serovars or serogroups and there are at least 200 known serogroup. Two serogroups, O1 and O139 have been associated with epidemic diseases. Virulence genes of these bacteria are OmpW, ctxA and tcpA.Objectives:Due to the importance of V. cholerae infection and developing molecular diagnostics of this organism in medical and microbiology sciences, this study aimed to describe molecular characterization of V. cholerae isolated from clinical samples using a molecular method.Materials and Methods:In this study, 48 samples were provided during summer 2013 (late August and early September) by reference laboratory. Samples were assessed using biochemical tests initially. The primer of OmpW, ctxA and tcpA genes was used in Polymerase Chain Reaction (PCR) protocols. Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR and Repetitive Extragenic Palindromic (REP)-PCR methods were used to subtype V. cholerae.Results:In this study, from a total of 48 clinical stool samples 39 (81.2 %) were positive for V. cholerae in biochemical tests and bacteria culture tests. The PCR results showed that of 39 positive isolates 35 (89.7%), 34 (87.1%) and 37 (94.8%) were positive for ctxA, tcpA and OmpW gene, respectively. Also, in the REP-PCR method with ERIC primer strains were divided into 10 groups. In the REP-PCR method with REP primer, strains were divided into 13 groups.Conclusions:Polymerase chain reaction has specificity and accuracy for identification of the organism and is able to differentiate biotypes. Enterobacterial repetitive intergenic consensus sequence is one of the informative and discriminative methods for the analysis of V. cholerae diversity. The REP-PCR is a less informative and discriminative method compared to other methods for the analysis of V. cholerae diversity.

Genotyping of Salmonella enterica subsp. enterica serovar Entritidis, isolated from poultry, cattle and human in Iran by ERIC-PCR

Salmonellosis is a serious disease in human and animals. One of the most important serovars (among more than 2610 known serovars of Salmonella) is Salmonella enterica serovar Entritidis which is a common foodborne pathogen worldwide. Various molecular subtyping techniques have been applied for identification of Salmonella. Enterobacterial repetitive intergenic consensus (ERIC) PCR techniques [collections of ERIC1R– ERIC2 primers] were examined for the discrimination of Salmonella enterica seovar Entritidis isolates at the serotype level. Sixty four S.Entritidis isolates from both human and (non-human) sources (cattle and poultry) in Iran, which previously were described by common culture, biochemical and serological procedures, were chosen. Enterobacterial repetitive intergenic consensus (ERIC) were selected for rep-PCR to generate DNA fingerprints for the S.Entritidis isolates. These banding patterns provided DNA fingerprints of different isolates. A dendogram was made by using NTSYS software, Version 2.0 and represented that most human isolates were separately clustered from the non-human isolates. This experiment also revealed that PCR fingerprinting with the ERIC primers is suitable for typing of the S.Entritidis isolates from different origin and for epidemiological trace back and taxonomy.

A proposal to unify two subspecies of Staphylococcus equorum: Staphylococcus equorum subsp. equorum and Staphylococcus equorum subsp. linens

Twelve isolates from jeotgal, a Korean high-salt-fermented seafood, identified as Staphylococcus equorum were compared by phenotypic and genotypic methods to determine their precise taxonomic identities at the subspecies level. Four strains and three strains had complete 16S rRNA gene sequence matches with S. equorum subsp. equorum DSM 20674(T) and S. equorum subsp. linens DSM 15097(T), respectively. Five strains showed 99.9 % identity with the sequences of both type strains. In our DNA-DNA hybridization analyses among two type strains and two isolates, the similarities were over 72 % and were higher than the similarities presented at the subspecies proposal. Physiological characteristics such as sugar utilization, β-galactosidase activity, novobiocin resistance and salt tolerance, which were adopted for subspecies separation, could not be applied to assign the isolates to a taxonomic unit. Antibiotic susceptibility, hemolytic activity, biofilm formation and protein profiles did not present markers to divide the isolates into either of the subspecies. Multilocus sequence typing of the sequences of the 16S rRNA gene and five housekeeping genes did not produce any coherent relationship among the isolates and type strains. Repetitive element-PCR fingerprinting using ERIC (enterobacterial repetitive intergenic consensus) primers classified 12 isolates to three genotypes, and the genotypes of both type strains coincided with two isolates expressing different characteristics. Based on these phenotypic and genotypic analyses results, we propose to unify the present two subspecies of S. equorum into one species, S. equorum.

Genetic variability of Iranian strains of Pseudomonas syringae pv. syringae causing bacterial canker disease of stone fruits

Strains of Pseudomonas syringae pv. syringae (Pss) were isolated from healthy and diseased stone fruits tissues sampled from 38 stone fruits orchard sites in Iran in 2010 and 2011. These strains were tested for pathogenicity and the presence of the syrB gene and were genetically characterized by using ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and IS50 (insertion sequences) primers and PCR. All 78 strains of Pss tested were moderately to highly pathogenic on Loring peach seedlings. A total of 78 isolates of the Pss amplified a 752-bp fragment with the syrB primers. To assess genetic diversity among the strains, genomic DNA was extracted from strains and used in rep-PCR and IS50-PCR analysis. Cluster analysis was performed using UPGMA. The strains of Pss were separated into nine distinguishable genotypic groups by the combination data set of both rep-PCR and IS50-PCR at 73 % similarity level. There was no significant correlation between genetic diversity and geographical origin of the isolates. These results indicate that a combination of rep-PCR and IS50-PCR fingerprinting can be used as a high resolution genomic fingerprinting method for elucidating intrapathovar diversity among strains of Pss. The results of this study demonstrated the existence of a considerable genetic diversity among Pss strains causing canker of stone fruit trees in Iran. In this study, genetic variability in Iranian strains of Pss were established, which will be of immense use in the development of resistant genotypes against this bacterial pathogen.

Rare occurrence of mupirocin resistance among clinical Staphylococcus isolates in Jordan

Staphylococcal infections have high occurrence in Jordanian patients. This study was carried out to determine the rates of high- and low-level mupirocin resistance (MupH and MupL) among staphylococci with the molecular characterization. Two hundred and thirty-two non-duplicate Staphylococcus spp. isolated from different clinical specimens were tested for mupirocin susceptibility using disk diffusion method and minimum inhibitory concentration (MIC). Resistance genes and clone relatedness was studied using polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus primers (Eric-PCR) for the latter. Plasmid curing was performed to determine the genetic location of MupA gene. Among the 232 strains, 144 (62%) were methicillin-resistant Staphylococcus aureus (MRSA), 33 (14.2%) methicillin-susceptible Staphylococcus aureus (MSSA) and 55 (23.7%) were of other coagulase-negative Staphylococcus spp. (CoNS). Of all strains tested, only 6 (2.6%) were mupirocin resistant. MecA gene was detected in both MupL and MupH strains but MupA gene was only detected in MupH. Plasmid curing improved the plasmidic location of MupA gene. Molecular typing by Eric-PCR method revealed heterogenicity of the genetic make up of our MupL and MupH strains. Staphylococci with MupA-carrying genes are present in Jordanian hospitals, but thank to the limited use of mupirocin, they remain rare.

Diversidade genética de isolados de Ralstonia solanacearum e caracterização molecular quanto a filotipos e sequevares

O objetivo deste trabalho foi identificar isolados brasileiros de Ralstonia solanacearum quanto a filotipos e sequevares, determinar sua diversidade genética, realizar a associação da estrutura genética do patógeno com sua classificação e origem geográfica e identificar um marcador molecular para a diagnose do moko-da-bananeira. Um grupo de 33 isolados de R. solanacearum, da coleção da Embrapa Tabuleiros Costeiros, coletado de diversos hospedeiros, foi caracterizado por meio de PCR em sequência palindrômica extragênica repetitiva (rep-PCR) e RAPD. Deste grupo, 19 perteciam à raça 2 do patógeno e 14 à raça 1, e 15 isolados eram associados à cultura da bananeira. Os filotipos e sequevares foram caracterizados e determinados por PCR Multiplex. Verificou-se que 82% dos isolados pertencem ao filotipo II, e 12% ao filotipo III. Todos os isolados da bananeira pertencem ao filotipo II. Atécnica de RAPD foi eficiente em agrupar os isolados de acordo com sua origem geográfica; entretanto, ela requer um número elevado de marcas moleculares. Foi possível relacionar os isolados pela análise rep-PCR. O iniciador com sequências repetitivas enterobacterianas intergênicas de consenso (ERIC) possibilitou a separação dos isolados de acordo com a raça, eoiniciador REP permitiua discriminação entre os filotipos. Estas duas análises foram as mais informativas.

GENETIC DIVERSITY OF XANTHOMONAS ORYZAE pv. ORYZAE STRAINS FROM RICE FIELDS IN MALAYSIA

Bacterial leaf blight caused by Xanthomonas oryzae pv oryzae (Xoo) is one of the most important bacterial diseases of rice, first detected in Japan in 1884. Repetitive extragenic palindromic (REP) and enterobacterial repetitive intergenic consensus (ERIC) assays were used to differentiate the phylogenetic relationships among 30 Xoo strains collected from rice fields in the states of Penang, Kedah, Selangor and Melaka (Peninsular Malaysia), during the period 2008-2010. Analysis of the 30 strains with REP and ERIC primers yielded five major amplification bands ranging from 200 to 800 bp in size. Fingerprints determined for each strain contained in total a maximum of 16 reproducible bands and a minimum of 9, ranging from 100 to 2,800 bp. The maximum number of score bands was observed in strains from Melaka and the minimum in strains from Penang and Kedah. Cluster analysis of the results of Rep PCRs yielded two major clusters and five sub clusters. Similarity between the two main clusters was 60% and 75% between five subclusters. This indicates that although there is a phylogenetic relation among strains of Xoo from rice crops of Peninsular Malaysia, nevertheless strains from different geographic regions are phylogenetically diverse.

An Outbreak of Post-Cataract Surgery Endophthalmitis Caused by Pseudomonas aeruginosa

Infectious endophthalmitis is among the most serious complications of cataract surgery. Gram-negative bacteria, including Pseudomonas aeruginosa, are responsible for less than 30% of cases; however, their rapidity of infection and virulence often results in poor visual outcome despite prompt antibiotic treatment. The purpose of this study was to investigate an outbreak of post-cataract surgery P. aeruginosa endophthalmitis in India. Hospital-based case series. Twenty patients with acute postoperative endophthalmitis who underwent cataract surgery at one of the peripheral centers of Joseph Eye Hospital, Tiruchirapalli, Tamil Nadu, India, from February 23 to April 2, 2008. Vitreous aspirates and environmental surveillance specimens were inoculated for culture. Antibiotic susceptibility testing was performed by agar diffusion method. Polymerase chain reaction (PCR) with enterobacterial repetitive intergenic consensus (ERIC) primers (ERIC-PCR) was used to establish the clonal relationship between clinical and environmental isolates. Post-cataract surgery P. aeruginosa endophthalmitis. Pseudomonas aeruginosa was isolated from 20 eyes with postoperative endophthalmitis, the phacoemulsifier's internal tubes, the povidone-iodine solution, and the operating theater air-conditioning system. All strains were multidrug-resistant to cefazolin, chloramphenicol, tetracycline, aminoglycosides, and fluoroquinolones; conversely, most of them were susceptible to polymyxin B. Polymerase chain reaction with enterobacterial repetitive intergenic consensus primers disclosed 2 major clusters: six genetically identical clinical isolates shared 94% of similarity with the air-conditioning isolate; 11 other clinical isolates had 88% of similarity with the former strain. Despite the prompt use of intravitreal antibiotics, 10 patients had evisceration or phthisis of the affected eye. The outcome of cataract surgery-related P. aeruginosa endophthalmitis is poor. The detection of multidrug-resistant isolates is a serious problem, jeopardizing an appropriate choice of treatment. Polymerase chain reaction with enterobacterial repetitive intergenic consensus results strongly suggest that the main source of infection in this outbreak was the contaminated air-conditioning system. Polymerase chain reaction with enterobacterial repetitive intergenic consensus is an inexpensive, fast, reproducible, and discriminatory DNA typing tool for effective epidemiologic surveillance of clinical and environmental isolates of P. aeruginosa.

Genetic variation ofPleiochaeta setosafromLupinus albus

A hierarchical collection of isolates of the fungal pathogen Pleiochaeta setosa was made from field-grown Lupinus albus plants across southern NSW, Australia in 2004. This collection, along with some historical reference isolates, was analysed for genetic variation using random amplified polymorphic DNA and enterobacterial repetitive intergenic consensus primer PCR. Anchored inter-simple sequence repeat primers were tested but produced little or no amplification or no detectable polymorphism. The observed overall variation was not explained by the hierarchical structure of the population (individual seedling, host species, host cultivar, regional location, or year). There was significant genetic variation between isolates even at the finest sampling scale (within a single seedling). Some very distinct individual isolates were identified. The high level of scale-independent genetic diversity seen in P. setosa suggests that field-based screening for Pleiochaeta root rotresistant genotypes in a L. albus breeding program at a single location is likely to result in resistance which is effective at multiple and widely separated locations. This resistance would have an increased chance of being polygenically controlled and durable under commercial cultivation conditions.

Genetic diversity among isolates of Paenibacillus larvae from Austria

Genetic diversity of 214 Paenibacillus larvae strains from Austria was studied. Genotyping of isolates was performed by polymerase chain reaction (PCR) with primers corresponding to enterobacterial repetitive intergenic consensus (ERIC), BOX repetitive and extragenic palindromic (REP) elements (collectively known as rep-PCR) using ERIC primers, BOX A1R and MBO REP1 primers. Using ERIC-PCR technique two genotypes could be differentiated (ERIC I and II), whereas using combined typing by BOX- and REP-PCR, five different genotypes were detected (ab, aB, Ab, AB and αb). Genotypes aB and αb are new and have not been reported in other studies using the same techniques.

Detection of Virulence Factors in Pseudomonas aeruginosa Strains Isolated From Contact Lens-Associated Corneal Ulcers

In microbial keratitis associated with contact lens use, Pseudomonas is the most common etiologic agent. The purpose of this study was to report on the microbiological findings of 8 P. aeruginosa strains isolated from contact lens-associated corneal ulcers. Scrapings from contact lens-related corneal ulcers were inoculated for culture. Identification and antibiotic susceptibility testing were performed by using the Vitek system (bioMérieux). The Pseudomonas' ability to form biofilm; produce gelatinase, elastase, and alkaline protease; and adhere to and invade human corneal epithelial cells was studied. Polymerase chain reaction with enterobacterial repetitive intergenic consensus primers (ERIC-PCR) was used to establish clonal relationship between the different isolates. All the strains showed multiple antibiotic resistance (resistance to 4 or more antibiotics), but all were susceptible to aminoglycosides and fluoroquinolones. Biofilm production was weak in 5 cases and absent in the remaining 3 cases. All isolates were able to produce alkaline protease and gelatinase but not elastase. Adherence to human corneal epithelial cells was poor (0-15 bacteria/cell) in 5 cases and medium (16-60 bacteria/cell) in 3 cases. Five isolates were found to be efficient invaders (>1000 CFU/mL). ERIC-PCR showed 8 different genetic patterns. Because multiresistant Pseudomonas isolates are common, we recommend antibiotic susceptibility testing in all cases of Pseudomonas keratitis so that, if there is no response to initial empiric treatment, antibiotics can be modified according to susceptibility results. The ability to produce alkaline protease and gelatinase and invade the corneal epithelium may play a major role in the pathogenesis of contact lens-related P. aeruginosa keratitis. Also, ERIC-PCR seems to be an inexpensive, fast, reproducible, and discriminatory DNA typing tool for effective epidemiologic surveillance of P. aeruginosa isolates potentially transmissible between patients with ocular infections.

Discrimination of Pseudomonas syringae isolates from sweet and wild cherry using rep-PCR

Bacterial canker is one of the most important diseases of cherry (Prunus avium). This disease can be caused by two pathovars of Pseudomonas syringae: pv. morsprunorum and pv. syringae. Repetitive DNA polymerase chain reaction-based fingerprinting (rep-PCR) was investigated as a method to distinguish pathovars, races and isolates of P. syringae from sweet and wild cherry. After amplification of total genomic DNA from 87 isolates using the REP (repetitive extragenic palindromic), ERIC (enterobacterial repetitive intergenic consensus) and BOX primers, followed by agarose gel electrophoresis, groups of isolates showed specific patterns of PCR products. Pseudomonas syringae pv. syringae isolates were highly variable. The differences amongst the fingerprints of P. syringae pv. morsprunorum race 1 isolates were small. The patterns of P. syringae pv. morsprunorum race 2 isolates were also very uniform, with one exception, and distinct from the race 1 isolates. rep-PCR is a rapid and simple method to identify isolates of the two races of P. syringae pv. morsprunorum; this method can also assist in the identification of P. syringae pv. syringae isolates, although it cannot replace inoculation on susceptible hosts such as cherry and lilac.

Identifying Host Sources of Fecal Pollution: Diversity of Escherichia coli in Confined Dairy and Swine Production Systems

Repetitive extragenic palindromic PCR fingerprinting of Escherichia coli is one microbial source tracking approach for identifying the host source origin of fecal pollution in aquatic systems. The construction of robust known-source libraries is expensive and requires an informed sampling strategy. In many types of farming systems, waste is stored for several months before being released into the environment. In this study we analyzed, by means of repetitive extragenic palindromic PCR using the enterobacterial repetitive intergenic consensus primers and comparative analysis using the Bionumerics software, collections of E. coli obtained from a dairy farm and from a swine farm, both of which stored their waste as a slurry in holding tanks. In all fecal samples, obtained from either barns or holding tanks, the diversity of the E. coli populations was underrepresented by collections of 500 isolates. In both the dairy and the swine farms, the diversity of the E. coli community was greater in the manure holding tank than in the barn, when they were sampled on the same date. In both farms, a comparison of stored manure samples collected several months apart suggested that the community composition changed substantially in terms of the detected number, absolute identity, and relative abundance of genotypes. Comparison of E. coli populations obtained from 10 different locations in either holding tank suggested that spatial variability in the E. coli community should be accounted for when sampling. Overall, the diversity in E. coli populations in manure slurry storage facilities is significant and likely is problematic with respect to library construction for microbial source tracking applications.

Intraspecies variability of Desulfovibrio desulfuricans strains determined by the genetic profiles

Fifteen (soil and intestinal) strains of Desulfovibrio desulfuricans species were typed by PCR method with the use of primers specific for repetitive extragenic palindromic (REP) and enterobacterial repetitive intergenic consensus (ERIC) sequences. As a result, characteristic DNA fingerprints for the strains were obtained. Moreover, the genetic profiles were found to be useful for typing and distinguishing the strains of D. desulfuricans. According to cluster analysis, PCR with primers complementary to the sequences REP appeared to be slightly more discriminatory than PCR with ERIC primers for the investigated strains. Distinct fingerprint patterns of two isolates derived from the same patient pointed to the different origin of both strains.

Clonality of Fusobacterium nucleatum in root canal infections.

Fusobacterium nucleatum is a gram-negative non-spore-forming, non-motile, obligate anaerobic rod that is normally isolated from the oral cavity. Several studies have reported a significant heterogeneity within the F. nucleatum species. The aim of the present study was to analyze the clonal diversity of F. nucleatum strains isolated from intracanal infections and to evaluate the presence of Enterobacterial Repetitive Intergenic Consensus (ERIC)-like sequences in the genome of F. nucleatum. Samples were collected from 13 single-root teeth from adult patients, all having carious lesions, necrotic pulps and radiographic evidence of periradicular bone loss. F. nucleatum was isolated from two different patients (subjects 5 and 7) by culture. Amplification of 19 colonies from subject 5 and 15 colonies from subject 7 using ERIC primers resulted in four clonal types, two per subject. An intense amplicon of approximately 700 bp was generated by ERIC-PCR for all F. nucleatum isolates and F. nucleatum ssp. polymorphum ATCC 10953. The amplification reaction using primer 1254 confirmed the results obtained with the ERIC primer. Our findings indicate that DNA fingerprints provided by ERIC- and Arbitrarily Primed (AP)-PCR may constitute a powerful tool for investigating F. nucleatum clonal diversity.

ERIC- and REP-PCR amplify non-repetitive fragments from the genome of Drechslera avenae and Stemphylium solani.

In the present study, the homology relationships among fragments amplified by ERIC (Enterobacterial Repetitive Intergenic Consensus) and REP (Repetitive Extragenic Palindromic) primers in Drechslera avenae and Stemphylium solani causing leaf blotch and black spot of oats and leaf blight of cotton, respectively, were investigated by Southern hybridization followed by DNA sequencing. ERIC- and REP-PCR were performed for 24 isolates of D. avenae and 33 isolates of S. solani. The banding patterns obtained were transferred to nylon membranes and a total of 16 fragments, four ERIC and four REP bands from each fungus, used as probes. The hybridization and DNA sequencing revealed a low degree of similarity among the bands indicating that the regions amplified by these primers are not repetitive and that the primers anneal at random regions in the genomes of D. avenae and S. solani.

ERIC- and REP-PCR amplify non-repetitive fragments from the genome of Drechslera avenae and Stemphylium solani

In the present study, the homology relationships among fragments amplified by ERIC (Enterobacterial Repetitive Intergenic Consensus) and REP (Repetitive Extragenic Palindromic) primers in Drechslera avenae and Stemphylium solani causing leaf blotch and black spot of oats and leaf blight of cotton, respectively, were investigated by Southern hybridization followed by DNA sequencing. ERIC- and REP-PCR were performed for 24 isolates of D. avenae and 33 isolates of S. solani. The banding patterns obtained were transferred to nylon membranes and a total of 16 fragments, four ERIC and four REP bands from each fungus, used as probes. The hybridization and DNA sequencing revealed a low degree of similarity among the bands indicating that the regions amplified by these primers are not repetitive and that the primers anneal at random regions in the genomes of D. avenae and S. solani.

Bacillus cereus keratitis associated with contact lens wear

Objective We report the first case of contact lens-related Bacillus cereus keratitis and ulcer associated with B. cereus contamination of the contact lens case. This is also the first study to investigate and establish the genetic identity of an organism isolated from the cornea and contact lens case in a patient with contact lens-associated keratitis. Design Case report. Intervention and testing Conjunctival swabs and corneal scrapings from the left eye were inoculated for culture. The contact lens case was also cultured. Antibiotic susceptibility testing was determined by agar disk diffusion method. Initial treatment with topical ciprofloxacin and fortified tobramycin was given. Genetic analysis of the bacterial isolates was performed using polymerase chain reaction (PCR) with enterobacterial repetitive intergenic consensus primers (ERIC; ERIC-PCR). Susceptibility of B. cereus to heat and contact lens disinfecting solutions containing hydrogen peroxide, hydrogen peroxide-catalase, polyquaternium-1, and polyaminopropyl biguanide (PAPB) was tested. Main outcome measures Clinical features, culture results, and antibiotic susceptibility testing were analyzed. The ERIC-PCR amplification products were visualized in ethidium bromide-stained agarose gel. Bacterial growth after exposure to heat and contact lens disinfecting solutions was assessed on blood agar plates. Results B. cereus was grown from the conjunctiva, corneal ulcer, and contact lens case. All isolates were sensitive to gentamicin, tobramycin, ciprofloxacin, clindamycin, and vancomycin. The corneal ulcer gradually healed over the next 6 days. Results of ERIC-PCR showed that the isolates from the cornea and contact lens case were indistinguishable, thus demonstrating the source of infecting organism to be the contaminated contact lens case. Exposure to a temperature of 80°C for 20 minutes and incubation with hydrogen peroxide-catalase, polyquaternium-1, and PAPB for the minimum recommended time failed to kill B. cereus. Only exposure to hydrogen peroxide for 4 hours eradicated the organism. Conclusions B. cereus should be considered a possible etiologic agent of contact lens-associated keratitis. Heat and many types of contact lens disinfecting solutions may be ineffective in eradicating B. cereus from contaminated contact lens cases. Only prolonged exposure to hydrogen peroxide appeared to be sporicidal to B. cereus in this study.