Prospective evaluation of an easy and reliable work flow for the screening of OXA-48-producing Klebsiella pneumoniae in endemic settings.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) represent a serious threat to public health. Clinical microbiology laboratories (CMLs) need effective protocols for screening and confirmation of CPE. AIM: To prospectively evaluate an algorithm for the screening of carbapenemase-producing Klebsiella pneumoniae in an OXA-48 endemic hospital. METHODS: The algorithm was based on a disc diffusion assay using ertapenem and temocillin, which also served as a purity check for routine automated antimicrobial susceptibility testing. All isolates with minimal inhibitory concentrations >0.5 mg/L or zone inhibition diameters <25 mm for ertapenem (Criterion 1) and <12 mm for temocillin (Criterion 2) were tested sequentially by an OXA-48 lateral flow immunochromatographic assay and a multiplex polymerase chain reaction targeting VIM, KPC and OXA-48. If neither test was positive, the modified Hodge test or CARBA NP test was used. FINDINGS: Over 2 years, 2487 K. pneumoniae were assessed by the algorithm proposed, and 378 (15.20%) met both criteria. Of these, 98.68% (373/378) were either confirmed as OXA-48 producers or originated from patients with a previous CPE isolate that maintained the same resistance phenotype over time. The remaining three K. pneumoniae were VIM producers. Only two of the 378 isolates (0.53%) did not produce carbapenemase, despite meeting Criteria 1 and 2. CONCLUSION: The algorithm described combined the most sensitive carbapenem for CPE detection with a cut-off for temocillin that was highly specific for detection of OXA-48. It is reliable and easy to apply in routine CML work flow, allowing rapid detection of CPE isolates and hence prompt implementation of infection control measures and targeted antimicrobial regimens.

Prospective multicentre study of rectal carriage of multidrug-resistant Enterobacteriaceae among health-care workers in Spain.

OBJECTIVES: To investigate the rectal carriage of multidrug-resistant Enterobacteriaceae (colistin-resistant, extended-spectrum ß-lactamase (ESBL) -producers and/or carbapenemase-producers) among health-care workers (HCWs) from six Spanish hospitals. METHODS: Rectal swabs from 258 HCWs, employed in intensive care units, haematology wards and clinical microbiology laboratories from six hospitals in northern Spain were studied. They were cultured in selective media for Gram-negative resistant bacteria. Detection of antimicrobial resistance genes and multilocus sequence typing were performed by PCR and further sequencing. A questionnaire including data related to risk factors of colonization/infection by resistant bacteria (age, gender, chronic diseases, immunosuppressive therapies, invasive procedures or antimicrobial treatments) was given to each participant. RESULTS: No carbapenemase-producing Enterobacteriaceae were recovered. However, 8/258 HCWs (3.1%) were positive for ESBL-producing isolates. This rate was not higher than the colonization rate previously reported in Spain for healthy people in the community. Five isolates showed high-level resistance to colistin (MICs ranging from 8 to 128 mg/L) but all of them were negative for the mcr genes tested. No statistically significant risk factors for gut colonization by ESBL-producing or colistin-resistant Enterobacteriaceae were identified among the HCWs participating in the study. CONCLUSIONS: Our data suggest that working in hospitals does not represent a risk for rectal carriage of multidrug-resistant Enterobacteriaceae.

Long-term endemic situation caused by a linezolid- and meticillin-resistant clone of Staphylococcus epidermidis in a tertiary hospital.

BACKGROUND: Linezolid (LZD)-resistant Staphylococcus epidermidis (LRSE) are increasing, and are mainly associated with outbreaks in hospital wards with high LZD consumption. AIM: To investigate the frequency of LRSE in a tertiary hospital in the context of LZD use. METHODS: The frequency of LRSE and the data on LZD usage [expressed as defined daily dose (DDD) per 100 patient-days], from 2011 to 2017, were analysed retrospectively. Selected LRSE were typed by pulsed-field gel electrophoresis (PFGE) and screened for transferable LZD resistance genes. Representative isolates were typed by multi-locus sequence typing, and ribosomal mechanisms of LZD resistance were investigated. FINDINGS: In total, 435 LRSE were detected, with frequencies ranging from 13.56% to 32.93% in the intensive care unit (ICU) where LZD consumption was high (6.34-8.10 DDDs), and from 2.48 to 6.80% in the remaining wards where LZD use was considerably lower (0.63-2.49 DDDs). The first 44 LRSE isolates recovered (June 2013-June 2014) were closely related according to PFGE patterns, and all except one were resistant to meticillin due to mecA production. Selected isolates belonged to ST2, carried SCCmec III, and had the G2576T mutation in the V domain of each of the six copies of the 23S rRNA gene. Five of the 44 isolates (11.36%) were positive for the cfr gene. CONCLUSION: An ST2 LZD- and meticillin-resistant clone was found in the ICU and also in wards with low consumption of LZD. This highlights the need to implement and maintain infection control measures as well as antimicrobial stewardship programmes in all hospital units in order to preserve the efficacy of LZD.

Nosocomial ventriculitis caused by a meticillin- and linezolid-resistant clone of Staphylococcus epidermidis in neurosurgical patients.

BACKGROUND: Postneurosurgical ventriculitis is mainly caused by coagulase-negative staphylococci. The rate of linezolid-resistant Staphylococcus epidermidis (LRSE) is increasing worldwide. AIMS: To report clinical, epidemiological and microbiological data from a series of ventriculitis cases caused by LRSE in a Spanish hospital between 2013 and 2016. METHODS: Cases of LRSE ventriculitis were reviewed retrospectively in a Spanish hospital over a four-year period. Clinical/epidemiological data of the infected patients were reviewed, the isolates involved were typed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing, and the molecular bases of linezolid resistance were determined. FINDINGS: Five cases of LRSE ventriculitis were detected. The patients suffered from cerebral haemorrhage or head trauma that required the placement of an external ventricular drain; spent a relatively long time in the intensive care unit (ICU) (10-26 days); and three out of the five patients had previously been treated with linezolid. All LRSE had the same PFGE pattern, belonged to ST2, and shared an identical mechanism of linezolid resistance. Specifically, all had the G2576T mutation in the V domain of each of the six copies of the 23S rRNA gene, together with the Q136L and M156T mutations and the 71GGR72 insertion in the L3 and L4 ribosomal proteins, respectively. CONCLUSION: The high ratio of linezolid consumption in the ICU (7.72-8.10 defined daily dose/100 patient-days) could have selected this resistant clone, which has probably become endemic in the ICU where it could have colonized admitted patients. Infection control and antimicrobial stewardship interventions are essential to prevent the dissemination of this difficult-to-treat pathogen, and to preserve the therapeutic efficacy of linezolid.

Evaluation of a real-time PCR assay for rectal screening of OXA-48-producing Enterobacteriaceae in a general intensive care unit of an endemic hospital.

Carbapenemase-producing Enterobacteriaceae are increasing worldwide. Rectal screening for these bacteria can inform the management of infected and colonized patients, especially those admitted to intensive care units (ICUs). A laboratory developed, qualitative duplex real-time polymerase chain reaction assay for rapid detection of OXA-48-like and VIM producing Enterobacteriaceae, performed on rectal swabs, was designed and evaluated in an intensive care unit with endemic presence of OXA-48. During analytical assay validation, no cross-reactivity was observed and 100% sensitivity and specificity were obtained for both blaOXA-48-like and blaVIM in all spiked clinical samples. During the clinical part of the study, the global sensitivity and specificity of the real-time PCR assay for OXA-48 detection were 95.7% and 100% (P=0.1250), respectively, in comparison with culture; no VIM-producing Enterobacteriaceae were detected. Clinical features of patients in the ICU who were colonized or infected with OXA-48 producing Enterobacteriaceae, including outcome, were analyzed. Most had severe underlying conditions, and had risk factors for colonization with carbapenemase-producing Enterobacteriaceae before or during ICU admission, such as receiving previous antimicrobial therapy, prior healthcare exposure (including long-term care), chronic disease, immunosuppression and/or the presence of an intravascular catheter and/or mechanical ventilation device. The described real-time PCR assay is fast (~2-3hours, if DNA extraction is included), simple to perform and results are easy to interpret, features which make it applicable in the routine of clinical microbiology laboratories. Implementation in endemic hospitals could contribute to early detection of patients colonized by OXA-48 producing Enterobacteriaceae and prevention of their spread.

Pseudomonas syringae pv. phaseolicola isolated from weeds in bean crop fields.

UNLABELLED: Pseudomonas syringae pv. phaseolicola, the causative agent of halo blight in common bean (Phaseolus vulgaris L.), was isolated from weeds associated with bean crops in Spain. The bacterium was recovered from Fumaria sp, Mercurialis annua, Solanum nigrum and Sonchus oleraceus. Ps. s. pv. phaseolicola had previously been isolated from leguminous plants and S. nigrum, but to our knowledge, this is the first time it was recovered from the other three species. The isolates were phenotypically and genetically characterized, and they were compared with isolates recovered from common beans. Five different genotypic profiles were detected by PmeI-PFGE, two of them being of new description. Weed isolates were as pathogenic on bean plants as bean isolates, but they were not pathogenic on S. nigrum. Regarding the survival of the pathogen in weeds, Ps. s. pv. phaseolicola was isolated from So. oleraceus 11 weeks after the end of the bean crop. These results strongly support the idea of weeds as a potential source of inoculum for halo blight in bean. SIGNIFICANCE AND IMPACT OF THE STUDY: It has traditionally been considered that the main source of inoculum of Pseudomonas syringae pv. phaseolicola causing halo blight disease in Phaseolus vulgaris are the bean seeds, and that the host range of the bacterium is almost restricted to leguminous plants. In this study, the bacterium was recovered from four nonleguminous weed species collected in bean fields, and its permanence in weeds for at least 11 weeks after the harvesting of the beans was demonstrated. We have also proved that the strains isolated from weeds were pathogenic on bean plants. Accordingly, the host range of Ps. s. pv. phaseolicola could be broader than previously thought and weeds appear to be acting as a reservoir of the pathogen until the next crop.

Detection and Molecular Characterization of Salmonella enterica Serovar Eppendorf Circulating in Chicken Farms in Tunisia.

Salmonella enterica subsp. enterica serovar Eppendorf, with antigenic formula 1,4,12,[27]:d:1,5, is an infrequent serovar. However, 14% (20 of 142) of the isolates recovered during June-July 2012 in chicken farms in Tunisia belonged to S. Eppendorf. These isolates were analysed for resistance and virulence profiles. None of them were susceptible to all antimicrobials tested, while 70%, 60%, 50%, 50%, 20% and 5% were resistant to sulphonamides (sul1, sul2 and sul3), streptomycin (aadA1-like), trimethoprim (dfrA1-like), nalidixic acid (GyrA Asp87 →Asn and not identified), gentamicin (not identified) and ampicillin (blaTEM -1-like). About 30% of the isolates showed decreased susceptibility to ciprofloxacin and carried the qnrB gene; 65% of the isolates were multidrug resistant and contained class 1 integrons with sul1 or sul3 in the 3' conserved segment. The orgA, ssaQ, mgtC, siiD and sopB virulence genes located on SPI1 to SPI5 and the fimbrial bcfC gene were present in all isolates; the sopE1 and sodC1 carried by prophages were variably detected; however, the prophage gipA gene and the spvC gene of serovar-specific virulence plasmids were absent. Altogether, ten resistance and three virulence profiles were identified. Typing of the isolates with XbaI- and BlnI-PFGE supports a close relationship, although they appear to be evolving under selective pressure probably caused by antimicrobial use in chicken husbandry. As far as we know, this is the first study investigating the molecular bases of antimicrobial drug resistance, the virulence gene content and the PFGE profiles of S. Eppendorf. The epidemiological surveillance of this serovar would be necessary to evaluate its possible impact on human health, particularly in Tunisia and other African countries where it was already reported.

Genotypes, exotoxin gene content, and antimicrobial resistance of Staphylococcus aureus strains recovered from foods and food handlers.

Staphylococcal food poisoning, one of the most common food-borne diseases, results from ingestion of one or more staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus in foods. In the present study, 64 S. aureus isolates recovered from foods and food handlers, associated or not associated with food-poisoning outbreaks in Spain, were investigated. They were assigned to 31 strains by spa typing, multilocus sequence typing (MLST), exotoxin gene content, and antimicrobial resistance. The strains belonged to 10 clonal complexes (CCs): CC5 (29.0%), CC30 (25.8%), CC45 (16.1%), CC8, CC15 (two strains each), CC1, CC22, CC25, CC59, and CC121 (one strain each). They contained hemolysin genes (90.3%); lukED (77.4%); exfoliatin genes eta, etd (6.5% each), and etb (3.2%); tst (25.8%); and the following enterotoxin or enterotoxin-like genes or clusters: sea (38.7%), seb (12.9%), sec (16.1%), sed-selj with or without ser (22.9%), selk-selq (6.5%), seh, sell, selp (9.7% each), egc1 (32.3%), and egc2 (48.4%). The number of se and sel genes ranged from zero to 12. All isolates carrying tst, and most isolates with genes encoding classical enterotoxins (SEA, SEB, SEC, and SED), expressed the corresponding toxin(s). Two CC5 isolates from hamburgers (spa type t002, sequence type 5 [ST5]; spa type t2173, ST5) were methicillin resistant and harbored staphylococcal cassette chromosome mec (SCCmec) IVd. Six (19.4%) were mupirocin resistant, and one (spa type t120, ST15) from a food handler carried mupA (MIC, 1,250 µg/ml). Resistance to ampicillin (blaZ) (61.3%), erythromycin (ermA-ermC or ermC) (25.8%), clindamycin (msrA-msrB or msrB) (16.1%), tetracycline (tetK) (3.2%), and amikacin-gentamicin-kanamycin-tobramycin (aphA with aacA plus aphD or aadD) (6.5%) was also observed. The presence of S. aureus strains with an important repertoire of virulence and resistance determinants in the food chain represents a potential health hazard for consumers and merits further observation.

Virulence and resistance determinants of German Staphylococcus aureus ST398 isolates from nonhuman sources.

A series of 100 Staphylococcus aureus isolates ascribed to sequence type 398 (ST398) and recovered from different sources (healthy carrier and diseased pigs, dust from pig farms, milk, and meat) in Germany were investigated for their virulence and antimicrobial resistance genetic background. Antimicrobial resistance was determined by the disk diffusion method. Virulence and resistance determinants (37 and 31 genes, respectively) were tested by PCR. Only two virulence profiles, including the accessory gene regulator agrI and three or four hemolysin-encoding genes, were detected. In contrast, 33 resistance profiles were distinguished (only 11 were shown by more than one isolate). Fifty-nine isolates were multiresistant (four or more antimicrobial classes), and 98 were methicillin resistant (mecA positive). All of the ST398 isolates showed resistance to tetracycline [encoded by tet(M) alone or together with tet(K) and/or tet(L)]. In addition, 98% were resistant to other antimicrobials, including macrolide-lincosamine-streptogramin B (70%, encoded by ermA, ermB, and ermC, alone or in combination), trimethoprim (65%, mostly due to dfrK and dfrG), kanamycin and gentamicin [29% and 14%, respectively, mainly related to aac(6')-Ie-aph(2″)-Ia and/or ant(4')-Ia but also to aph(3')-IIIa], chloramphenicol (9%, fexA or cfr), quinupristin-dalfopristin (9%), ciprofloxacin (8%), and trimethoprim-sulfamethoxazole (4%). The heterogeneity of the resistance profiles underlines the ability of the ST398 clone to acquire multiple antimicrobial resistance genes. However, the virulence gene content of the tested isolates was low. Continuous surveillance is needed to clarify whether its pathogenicity potential for animals and humans will increase over time.

Spread of a multiresistant CTX-M-9-producing Salmonella enterica serotype Virchow phage type 19 in Spain.

The purpose of this study was to survey Salmonella enterica serotype Virchow phage type 19 (S. Virchow PT19) strains submitted to the Spanish National Reference Laboratory for Salmonella (SNRLS) from 2002 to 2006 in order to determine the rate type and genetic background of beta-lactam resistance and to further identify the associated resistances. Ninety-nine S. Virchow PT19 strains were analysed. Antimicrobial susceptibility was determined by the disk diffusion method using Mueller-Hinton agar medium. Polymerase chain reaction (PCR) assays and, later, sequencing of the obtained fragments were performed for the molecular characterisation of the resistances. Pulsed-field gel electrophoresis (PFGE) and plasmid analysis (using conjugation, Southern blot hybridisation and replicon typing) were used for characterisation. The characterisation of S. Virchow PT19 strains allowed the identification of a clonal multiresistant S. Virchow PT19 harbouring an IncH12 plasmid with the bla (CTX-M-9) gene within the complex integron In60 distributed across Spain. An IncH12 plasmid widely reported and studied in Enterobacteria is described in a clonal multiresistant S. Virchow PT19 which has successfully spread throughout Spain.

The emerging methicillin-resistant Staphylococcus aureus ST398 clone can easily be typed using the Cfr9I SmaI-neoschizomer.

AIM: To establish a PFGE protocol using Cfr9I, neoschizomer of SmaI, for typing of Staphylococcus aureus isolates belonging to the emerging MRSA ST398 clone. METHODS AND RESULTS: Staphylococcus aureus ST398 and non-ST398 isolates were analysed using the PFGE conditions recommended by the HARMONY consensus protocol. Genomic DNA of non-ST398 isolates could be digested with SmaI, XmaI (also a SmaI-neoschizomer) and Cfr9I. The DNA of SmaI-nontypeable ST398 isolates was partially resistant to XmaI, but could be digested with Cfr9I. By PCR-amplification/sequencing, the presence of a novel C5-cytosine methyltransferase gene (sauST398M) was detected in the ST398 isolates. The encoded enzyme, which shows high similarity with C5-cytosine methyltransferases that modify the CCCGGG recognition sequence, could be responsible for the different restriction results. CONCLUSION: SmaI-PFGE is regarded as the 'gold standard' for typing S. aureus. Because of different susceptibility of the GGGCCC recognition sites of the ST398 DNA against SmaI, XmaI and Cfr9I, the proposed protocol is a valuable tool for ST398 typing. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of this protocol allows the comparison of results from SmaI-nontypeable isolates with S. aureus SmaI-PFGE databases and can be applied for outbreak investigations and traceability studies of this emerging MRSA clone.

High heterogeneity within methicillin-resistant Staphylococcus aureus ST398 isolates, defined by Cfr9I macrorestriction-pulsed-field gel electrophoresis profiles and spa and SCCmec types.

During recent years, the animal-associated methicillin-resistant Staphylococcus aureus clone ST398 has extensively been studied. The DNA of these isolates turned out to be refractory to SmaI restriction, and consequently, SmaI is unsuitable for subtyping this clone by standard pulsed-field gel electrophoresis (PFGE). Very recently, ST398 DNA was shown to be digested by Cfr9I, a neoschizomer of SmaI. In the present study, we employed Cfr9I PFGE on 100 German and 5 Dutch ST398 isolates and compared their PFGE profiles, protein A gene variable repeat regions (spa types), and types of the staphylococcal cassette chromosome mec (SCCmec). The isolates (from healthy carrier pigs, clinical samples from pigs, dust from farms, milk, and meat) were assigned to 35 profiles, which were correlated to the SCCmec type. A dendrogram with the Cfr9I patterns assigned all profiles to two clusters. Cluster A grouped nearly all isolates with SCCmec type V, and cluster B comprised all SCCmec type IVa and V* (a type V variant first identified as III) carriers plus one isolate with SCCmec type V. Both clusters also grouped methicillin-susceptible S. aureus isolates. The association of the majority of isolates with SCCmec type V in one large cluster indicated the presence of a successful subclone within the clonal complex CC398 from pigs, which has diversified. In general, the combination of Cfr9I PFGE with spa and SCCmec typing demonstrated the heterogeneity of the series analyzed and can be further used for outbreak investigations and traceability studies of the MRSA ST398 emerging clone.

Detection of Salmonella enterica serovar Typhimurium with pUO-StVR2-like virulence-resistance hybrid plasmids in the United Kingdom.

The aim of this study was to investigate the presence in the United Kingdom (UK) of Salmonella enterica serovar Typhimurium isolates carrying pUO-StVR2-like virulence-resistance hybrid plasmids that originated from pSLT. One hundred and fifty ampicillin-resistant isolates of S. Typhimurium, collected in different regions of the UK during 2006, were screened for the presence of bla (OXA-1) carried by an InH-like integron (2000 bp/bla (OXA-1)-aadA1) characteristic of pUO-StVR2. Positive isolates were tested for the presence of a large plasmid that hybridised with probes specific for the bla (OXA-1) and spvC genes, used as resistance and virulence markers of the hybrid plasmid, respectively. Eleven out of the 150 isolates fulfilled both criteria and were assigned to the S. Typhimurium pUO-StVR2 group. Nine were resistant to ampicillin, chloramphenicol, streptomycin/spectinomycin, sulfonamides and tetracycline, encoded by bla (OXA-1), catA1, aadA1-like, sul1 and tet(B), respectively, and carried a pUO-StVR2-like plasmid of ca. 130 kb. Two contained hybrid plasmids of smaller size and lacked resistance(s) to chloramphenicol or chloramphenicol and tetracycline. The eleven isolates, which showed five and six closely related XbaI and BlnI profiles, respectively, were resistant to nitrofurantoin. In conclusion, multidrug-resistant S. Typhimurium isolates of the pUO-StVR2 group, which are endemic in Spain, were also detected in the UK, albeit with a low frequency (7.3%).

Clonal complexes and diversity of exotoxin gene profiles in methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates from patients in a Spanish hospital.

Molecular epidemiology studies have allowed the identification of the methicillin (meticillin)-resistant (MRSA) and methicillin-susceptible (MSSA) clonal complexes (CCs) and clones of Staphylococcus aureus circulating in a Spanish hospital recently. Of 81 isolates tested, 32.1% were MRSA. Most of them carried staphylococcal cassette chromosome mec (SCCmec) IVc (88.5%) and belonged to CC5 (88.5%; multilocus sequence typing types ST125 [mainly associated with spa type t067], ST5, and ST228). A higher diversity was found among MSSA isolates (67.9%). Eighty percent shared the genetic background of major MRSA lineages (CC5 [38.2%; ST125 and ST5], CC30 [25.5%; ST30], CC45 [14.5%; ST45 and ST47], and CC8 [1.8%; ST8]), but CC12, CC15, CC51, and CC59 were also detected. Many exotoxin genes were present in each of the 81 isolates, independent of whether they were involved in sepsis (11 to 22) or other types of infections (13 to 21), and they appeared in 73 combinations. The relevant data are that (i) all isolates were positive for hemolysin and leukotoxin genes (98.8% for lukED and 25.9% for lukPV); (ii) all contained an enterotoxin gene cluster (egc with or without seu), frequently with one or more genes encoding classical enterotoxins; (iii) about half were positive for tst and 95% were positive for exfoliatin-encoding genes (eta, etb, and/or etd); and (iv) the four agr groups were detected, with agrII (55.6%) and agrIII (23.5%) being the most frequent. Taken together, results of the present study suggest a frequent acquisition and/or loss of exotoxin genes, which may be mediated by efficient intralineage transfer of mobile genetic elements and exotoxin genes therein and by eventual breakage of interlineage barriers.

Erwinia persicina Causing Chlorosis and Necrotic Spots in Leaves and Tendrils of Pisum sativum in Southeastern Spain.

In 2003, symptoms of generalized chlorosis as well as necrosis in leaves and tendrils were observed in Pisum sativum L. cv Tirabeque grown in green fields in southeastern Spain (Granada Province), and by 2004, the disease affected approximately 12 ha. Bacteria isolated from symptomatic samples were gram negative, rod shaped, motile, oxidase negative, facultatively anaerobic, and fermentative, which coincided with the general characteristics of the family Enterobacteriaceae. The gene encoding the 16S rRNA from two isolates (LPPA 406 and LPPA 408) was sequenced after PCR amplification (1). The two sequences were identical (EMBL Accession No. AM294946 for LPPA 408) and showed 99% similarity with several strains of Erwinia persicina (including the type strain ATCC 35998, LPPA 373, LMG 11254, GS04, and LMG 2691). Additional biochemical tests were performed using E. persicina ATCC 49742 as a control. The three strains were negative for arginine dihydrolase activity, indol production, hydrolysis of casein, and hydrolysis of gelatin. In contrast, they were positive for assimilation of adonitol, l-lactate, mannitol, m-inositol, erythritol, sorbitol, sucrose, nitrate reduction, hydrolysis of aesculin, and growth in 5% NaCl at 36°C. Nevertheless, E. persicina ATTC 49742, but not the isolates from P. sativum, produced a pink pigment. The latter isolates were also tested for pathogenicity. Bacterial suspensions (108 CFU/ml) were spray inoculated on 10 pea seedlings of cv. Tirabeque. Seedlings were covered with transparent plastic bags for 2 days and held in an incubation chamber at 22°C and 80% relative humidity with a 12-h photoperiod. Assays were conducted twice. Symptoms that developed were similar to those originally observed in the field, whereas symptoms did not occur on control seedlings sprayed with sterile distilled water. Bacteria sharing the characteristics of the inoculated isolates were recovered from symptomatic plants, hence fulfilling Koch's postulates. E. persicina has been isolated previously from bean in the United States (3) and southeastern Spain (1) and from tomato, banana, and cucumber in Japan (2). To our knowledge, this is the first report of the bacterium on P. sativum. References: (1) A. J. González et al. Plant Dis. 89:109, 2005. (2) M. V. Hao et al. Int. J. Syst. Bacteriol. 40:379, 1990. (3) M. L. Schuster et al. Fitopatol. Bras. 6:345, 1990.

Molecular epidemiology of emergent multidrug-resistant Salmonella enterica serotype Typhimurium strains carrying the virulence resistance plasmid pUO-StVR2.

OBJECTIVES: To evaluate the incidence of a distinct multidrug-resistant (MDR) grouping of Salmonella serotype Typhimurium strains carrying the hybrid virulence resistance plasmid pUO-StVR2, and its possible evolution in the region where it was first detected [Principality of Asturias (PA), Spain]. METHODS: pUO-StVR2-containing isolates were tentatively identified by two genetic markers: the bla(OXA-30) gene and the class 1 integron InH:2000 bp/bla(OXA-30)-aadA1a. Positive isolates were examined for resistance profile (RP), plasmid content, virulence profile (VP) and genomic polymorphisms using macrorestriction-PFGE. RESULTS: A total of 182 out of 248 Typhimurium clinical isolates recorded in the PA over 2001-02 were ampicillin-resistant and could be distributed into several MDR groupings. A MDR grouping carrying pUO-StVR2, with a defined RP (AMP/bla(OXA-30), CHL/catA1, [STR-SPT]/[strA/B,aadA1a], SUL/[sul1,sul2], TET/tet(B), qacEDelta1, merA, +/-TMP/dfrA12, and containing InH), was represented by 49 isolates. The VPs of these isolates (24 genes screened) differed from that of the type strain LT2 by the absence of the sopE1 and pef genes. Macrorestriction analysis established six combined XbaI/BlnI PFGE profiles, and supported a clonal relationship among most of the isolates. CONCLUSIONS: During 2001-02, the isolates carrying pUO-StVR2 constituted the second most frequent S. Typhimurium MDR grouping recorded in the PA, preceded only by the pandemic pentaresistant DT104. Polymorphisms on the genomic DNA, different phage types, different plasmid profiles and the detection of trimethoprim resistance in one isolate encoded by an additional plasmid, were consistent with both intra-cluster evolution and horizontal transfer of the hybrid plasmid.

Pseudomonas viridiflava Causing Necrotic Leaf Spots and Defoliation on Hebe spp. in Northern Spain.

Hebe spp. is gaining interest as an ornamental crop in Spain. In October of 2003, plants of Hebe spp. (cv. Pink Paradise, Recurva, and Topiaria) grown in a nursery located in a northern region of Spain (Principality of Asturias) developed dark reddish spots in leaves. The spots (favored by high-humidity conditions) appeared initially in internal and lower leaves, but eventually progressed to the rest of the plant causing extensive defoliation. Samples from symptomatic leaves were processed for microbiological analysis. From the samples, Stemphylium sp. and fluorescent Pseudomonas sp., identified by conventional microbiological methods (1,3), were consistently recovered. Two isolates of the fungi and two of the bacterium (LPPA 365 and LPPA 366) were used in artificial inoculations of healthy plants (cv. New Zeeland) and all were performed in duplicate. A Stemphylium sp. was inoculated by irrigation of four seedlings with a suspension obtained by homogenization of the mycelium collected from two plates of potato dextrose agar in 100 ml of sterile distilled water. An equal number of seedlings were sprayed with a bacterial suspension (108 CFU/ml) in yeast extract peptone glucose broth. The same methods were used to inoculate all four combinations of fungi and bacteria. The seedlings were maintained at 22°C during 1 month with a 16/8-h photo-period, but in inoculations with bacteria they were initially kept under transparent plastic bags for 48 h to facilitate entry. Seedlings treated with sterile distilled water were included as controls. Although a powdery film of sooty mold was observed in some of the leaves irrigated with one of the two Stemphylium sp. strains, rusty lesions and defoliation did not occur on plants inoculated with the fungal isolates alone. In contrast, symptoms comparable with those observed in the field developed in plants inoculated with the bacteria, either alone or together with the fungus. Reisolation of fluorescent Pseudomonas sp. from symptomatic leaves taken from inoculated plants confirmed pathogenicity on Hebe spp. The pathogenic isolates were tentatively identified as atypical Pseudomonas viridiflava on the basis of biochemical tests (2,3), and their identity was then confirmed by sequencing of the gene encoding the 16S rRNA (3; GenBank Accession Nos. AM182933 and AM182934 for LPPA 365 and 366, respectively). To our knowledge, this is the first report of P. viridiflava as the cause of necrotic leaf spots and defoliation in Hebe spp. References: (1) H. L. Barnett and B. B. Hunter. Illustrated Genera of Imperfect Fungi. 3rd ed. Burgess Publishing Co, Minneapolis, MN, 1972. (2) A. J. González et al. Appl. Environ. Microbiol. 69:2936, 2003. (3) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470. 1966.

Genetic basis of antimicrobial drug resistance in clinical isolates of Salmonella enterica serotype Hadar from a Spanish region.

The genetic bases of antimicrobial drug resistance (R) of 79 Salmonella enterica serotype Hadar clinical isolates (recovered during 1995-2001 in a Spanish region) was investigated. The isolates showed a limited genomic variation, as demonstrated by PFGE analysis using XbaI (three profiles, S>or=0.77) and BlnI (seven profiles, S>or=0.49; with 95% of the isolates falling into two clusters, S>or=0.75). Thirteen R-profiles, ranging from susceptible to multidrug resistant, were recognized. All susceptible isolates (14%) were recovered before or during 1998, when multidrug resistance (MDR) was still uncommon (20% from 1995-1998). In later years, the percentage of MDR increased considerably (92% in 2001). Resistance to nalidixic acid, tetracycline, streptomycin and ampicillin-cefalotin, encoded by gyrA-Asp87/Asn, tet(A), strA/B, and bla (TEM) genes, respectively, were the most common, appearing together in 38% of the isolates. In all tetracycline- and streptomycin-resistant isolates, strA/B and tet(A) were chromosomally located, whereas bla (TEM) was plasmid-born. Five different bla (TEM) plasmids (pUO-ShR1 to pUO-ShR5, of about 9.4, 23, 30, 45, and 95 kb, respectively) were identified. pUO-ShR3 and pUO-ShR5 harbored additional R-genes: [dfrA1] and [acc(3)IV-strA/B], respectively. pUO-Sh2, pUO-Sh3, pUO-ShR4, and pUO-Sh5 were self-transferable, and the latter could also mobilize pUOShR1. The reported data constitute a useful background for further epidemiological studies of MDR in S. Hadar.

Cytotoxin and pyrogenic toxin superantigen gene profiles of Staphylococcus aureus associated with subclinical mastitis in dairy cows and relationships with macrorestriction genomic profiles.

A set of 84 Staphylococcus aureus isolates collected from the milk of cows with subclinical mastitis in Asturias (a cattle region of Spain) and six control strains were tested for sequences of genes encoding hemolysins (hla, hlb, hld, hlg, and hlg-2), leukotoxins (lukPV, lukM, and lukED), toxic shock syndrome toxin (tst), and enterotoxins (sea to see, seg to ser, and seu) by conventional and multiplex PCR. It was found that 84, 83, 11, and 39 isolates carried some type of hl, luk, tst, or se gene, respectively, which were arranged in 14 exotoxin genotypes. All of the isolates were negative for lukPV, hlg, sea, sed, see, sej, sek, sep, seq, and ser. Two gene groupings could be related with pathogenicity islands-[lukED, seg, sei, sem, sen, seo +/- seu] with Sabeta-1 and [tst, sec, sel] with SaPIbov, present in 45 and 13.1% of the isolates, respectively-while 11.9% of them carried both islands. Only one contained seb (together with upsilonSabeta-1), and another contained seh (together with lukED). The isolates were also analyzed by pulsed-field gel electrophoresis performed with SmaI. Thirty-nine SmaI profiles (similarity coefficient [S] = 0.94 to 0.21) were differentiated; 12, 1, and 10 of these, respectively, were generated by isolates presumptively carrying Sabeta-1, SaPIbov, or both. Five SmaI profiles (S > or = 0.8) formed a cluster, which contained 20 and 10 isolates carrying one (upsilonSabeta-1) or both islands. These data show the high frequency of genes encoding cytotoxins and pyrogenic toxin superantigens, their relationship with pathogenicity islands, and their distribution among a diversity of genetic types of S. aureus related to subclinical mastitis.