Role of IncHI2 plasmids harbouring blaVIM-1, blaCTX-M-9, aac(6')-Ib and qnrA genes in the spread of multiresistant Enterobacter cloacae and Klebsiella pneumoniae strains in different units at Hospital Vall d'Hebron, Barcelona, Spain.

Seven Enterobacter cloacae isolates and seven Klebsiella pneumoniae isolates harbouring a phenotype compatible with the production of a metallo-ß-lactamase were recovered between 2009 and 2011 in three Intensive Care Units of Hospital Vall d'Hebron (Barcelona, Spain). The presence of bla(VIM), bla(IMP), bla(NDM), bla(CTX-M), aac(6')-Ib, qnrA, qnrB and qnrS genes was screened by polymerase chain reaction (PCR) and sequencing. Clonal relatedness of the isolates was assessed by pulsed-field gel electrophoresis (PFGE) and, in the case of K. pneumoniae isolates, by multilocus sequence typing (MLST). PCR-based replicon typing, Southern hybridisation, plasmid double-locus sequence typing and MOB relaxase classification methods were used to identify and characterise the plasmids carrying the resistance genes. Transferability of the identified plasmids was tested by conjugation assays. All 14 isolates were found to carry bla(VIM-1), bla(CTX-M-9) (except one isolate), aac(6')-Ib and qnrA genes. Clonality assessment demonstrated that E. cloacae isolates were distributed in three clonal clusters, whereas all of the K. pneumoniae isolates belonged to one unique clone, identified as sequence type ST252. All studied isolates harboured a large conjugative IncHI2 MOB(H11) plasmid carrying all of the detected resistance genes. Plasmid DNA analysis showed that all of them belonged to the ST1 IncHI2 plasmid cluster and shared the same relaxase partial sequence. In conclusion, the present study describes the dissemination within a hospital of multiresistant E. cloacae and K. pneumoniae isolates producing VIM-1. A complex clonal epidemiology of the E. cloacae isolates was observed and plasmid DNA analysis strongly supports horizontal exchanges of the same IncHI2 plasmid between different strains and species.

First detection of plasmid-encoded blaOXY beta-lactamase.

Three Klebsiella oxytoca isolates and one Klebsiella pneumoniae isolate from three children admitted to the Hematology Unit of Hospital Vall d'Hebron (Barcelona, Spain) exhibited a susceptibility pattern suggesting OXY beta-lactamase hyperproduction. All the isolates contained a 95-kb plasmid that harbored bla(OXY-1), which was transferred by electrotransformation but could not be self-transferred by conjugation. A qnrS1 gene was also harbored in the bla(OXY-1)-carrying plasmid. This is the first report of a plasmid-encoded OXY beta-lactamase.

Dissemination of extended-spectrum beta-lactamase-producing bacteria: the food-borne outbreak lesson.

OBJECTIVES: Commensal and opportunistic bacteria producing extended-spectrum beta-lactamases (ESBL-PB) have undergone a broad and rapid spread within the general population; however, the routes of dissemination have not been totally elucidated. The aim of this study was to determine whether individuals involved in an outbreak of acute gastroenteritis, in addition to the enteropathogenic microorganism, share an ESBL-PB as indirect demonstration of its transmission from a common food source. METHODS: From 2003 to 2004 in Barcelona, Spain, stool samples from 905 people involved in 132 acute gastroenteritis outbreaks and 226 food handlers related to the outbreaks were investigated. RESULTS: In 31 outbreaks, 58 diners carrying one or more ESBL-PB were detected. In 10 outbreaks, two or more diners shared the same ESBL-PB, and in four of them, the strain was shared with the food handlers. CONCLUSIONS: This study provides circumstantial evidence that foods can be a transmission vector for ESBL-PB, probably from two reservoirs, food animals and food handlers.

Prevalence of qnr genes among extended-spectrum beta-lactamase-producing enterobacterial isolates in Barcelona, Spain.

OBJECTIVES: To evaluate the presence of qnr genes among enterobacterial isolates carrying extended-spectrum beta-lactamases (ESBLs) in Barcelona, Spain. METHODS: Screening for the qnrA, qnrB and qnrS genes was carried out by PCR amplification with specific primers in 305 non-duplicate, clinically relevant ESBL-producing enterobacterial isolates obtained from February 2003 to August 2004. ESBLs from all qnr-positive isolates were characterized by isoelectric focusing, PCR amplification and DNA sequencing. Plasmid analysis was performed by S1 digestion and hybridization with specific probes for the qnr and bla genes. Plasmids containing qnr genes were transferred by conjugation or transformation. The genetic environment of qnrA1 in selected isolates was characterized by cloning experiments. RESULTS: Fifteen isolates, each from a different individual, carried qnr. Among them, 14 had qnrA1 (6 Klebsiella pneumoniae, 6 Enterobacter cloacae and 2 Escherichia coli isolates) and 1 had qnrS1 (K. pneumoniae). None of the isolates carried qnrB. Among the qnrA1-carrying isolates, 10 possessed both bla(CTX-M-9) and bla(SHV-12), 2 had both bla(CTX-M-9) and bla(SHV-92) and 2 had bla(CTX-M-9) alone. The isolate with qnrS1 possessed bla(SHV-12). The qnrA1 and ESBL genes were located together on plasmids ranging in size from 40 to 320 kb. qnrS1 and bla(SHV-12) were not located on the same plasmid. Transfer of quinolone resistance was successfully achieved from all but three isolates. The cloned region surrounding qnrA in two K. pneumoniae isolates revealed a novel genetic organization. CONCLUSIONS: The prevalence of qnr among enterobacterial clinical isolates carrying ESBLs between 2003 and 2004 in Barcelona was 4.9%. qnrA1 was the most prevalent, whereas only one qnrS and no qnrB were detected.

Pathogenicity island markers in commensal and uropathogenic Escherichia coli isolates.

Uropathogenic isolates of Escherichia coli (UPEC) contain blocks of DNA, termed pathogenicity islands (PAIs), that contribute to their virulence. Two multiplex PCR assays were developed to detect eight PAI markers among 50 commensal E. coli and 100 UPEC isolates. In total, 40% of commensal isolates and 93% of UPEC carried PAIs. Despite this difference, the distribution of various PAIs showed the same pattern in both groups, with the most prevalent being PAI IV(536) (38% commensal vs. 89% UPEC), followed by PAI I(CFT073) (26% vs. 73%), PAI II(CFT073) (14% vs. 46%), PAI II(J96) (8% vs. 34%), PAI I(536) (8% vs. 33%) and PAI II(536) (4% vs. 20%). PAI III(536) was detected only in UPEC (2%), while PAI I(J96) was not detected in any isolate. Although the mean number of PAIs per isolate was higher among UPEC (2.97) than in commensal (0.98) isolates, there were no statistical differences among group B2 E. coli from the two origins; however, commensal isolates from groups D and B1 appeared to be less virulent than pathogenic isolates. Regardless of their phylogenetic group, nearly all the commensal and UPEC isolates with the same number of PAIs had the same PAI combinations. Although group B2 E. coli are uncommon among commensal intestinal flora, they are highly virulent when present, suggesting that the intestinal flora may act as a reservoir for bacteria that can cause urinary tract infection.

In vivo reversion to the wild-type beta-lactam resistance phenotype mediated by a plasmid carrying ampR and qnrA1 in Enterobacter cloacae.

Resistance to beta-lactams and quinolones in two isogenic Enterobacter cloacae isolates was studied. One was susceptible to cefoxitin and amoxicillin-clavulanate. The other one showed its natural beta-lactam resistance pattern. Both isolates had a nonfunctional AmpR regulator. However, within the second one, the presence of a plasmid carrying ampR and qnrA1 allowed reversion to the wild-type beta-lactam resistance phenotype and decreased susceptibility to fluoroquinolones.

[Patterns of susceptibility to antibiotics of Enterobacteriaceae causing intra-abdominal infection in Spain: SMART 2003 study outcomes]. / Patrones de sensibilidad a antimicrobianos de Enterobacteriaceae causantes de infecciones intraabdominales en España: resultados del estudio SMART 2003.

SMART (Study for Monitoring Antimicrobial Resistance Trends) is an ongoing global antimicrobial surveillance program focused on clinical isolates from intra-abdominal infections. The objective of this subanalysis was to assess antimicrobial susceptibility patterns among Entero-bacteriaceae recovered at 13 participating Spanish sites during 2003. Antimicrobial susceptibility testing was performed using broth microdilution techniques according to the CLSI (formerly NCCLS) guidelines for MIC testing. The presence of extended-spectrum beta-lactamases (ESBL) was confirmed in isolates with a MIC of ceftriaxone, ceftazidime, or cefepime>or=2 mg/l by comparing cefepime MICs with and with-out clavulanate. A total of 981 Enterobacteriaceae recovered from 840 patients were tested, of which 398 (41%) were community-acquired. Escherichia coli was the most common isolate (571 isolates; 58%), followed by Klebsiella spp. (153; 16% Enterobacter spp. (97; 10%), and Proteus spp. (63; 6%). A total of 191 isolates (19%) from 176 patients produced inducible beta-lactamases. The carbapenems and amikacin were the most consistently active agents against the Enterobacteriaceae (susceptibility>or=99%). Resistance rates for ceftazidime, cipro-floxacin, and levofloxacin exceeded 10%. ESBLs were detected phenotypically in 61 (6%) isolates, being the most common E. coli (61%), Klebsiella spp. (20%), and Enterobacter spp. (8%). Antimicrobial resistance among Enterobacteriaceae isolated from intra-abdominal infections is a problem in Spain. A significant proportion of inducible beta-lactamase and ESBL-producing Enterobacteriaceae causing intra-abdominal infection were acquired in the community. The carbapenems ertapenem, imipenem and meropenem and the aminoglycoside amikacin were highly active in vitro against Enterobacteriaceae isolated from intra-abdominal sites, including ESBL-producing organisms.

Relationship between Escherichia coli strains causing urinary tract infection in women and the dominant faecal flora of the same hosts.

To clarify whether prevalence or special pathogenicity is more important in determining urinary tract infection (UTI) causation, we compared the biotype, phylogenetic group, and virulence genes of Escherichia coli urine strains from 11 women with acute lower UTI with those of the host's dominant intestinal E. coli strain(s). Twenty-one unique E. coli clones were identified. For three women, the single faecal clone identified was also the host's urine clone, whereas for eight women faecal samples yielded 1 or 2 distinct non-urine clones (total, n = 10), either with (n = 3) or without (n = 5) the concurrent urine clone. The eight urine clones from the latter eight women exhibited significantly greater inferred virulence, according to virulence gene content and phylogenetic background, than did the hosts' 10 corresponding 'faecal only' clones. In contrast, the three urine clones that were detected as the host's sole faecal clone exhibited significantly lower inferred virulence than the other eight urine clones, and were statistically indistinguishable from the 10 'faecal only' clones. In conclusion, special pathogenicity is an important determinant of UTI pathogenesis in women, although prevalence may occasionally allow less virulent strains to cause UTI.

Molecular epidemiology of caliciviruses causing outbreaks and sporadic cases of acute gastroenteritis in Spain.

The molecular epidemiology of human caliciviruses (HuCVs) causing sporadic cases and outbreaks of acute gastroenteritis around eastern Spain (Catalonia and the Valencian Community) was studied by reverse transcription-PCR (RT-PCR) and by sequencing part of the RNA polymerase gene in open reading frame 1. HuCVs were detected in 44 of 310 stool specimens (14.19%) negative for other enteric pathogens obtained from children with acute gastroenteritis. Norwalk-like viruses (NLVs) were the most common cause of the gastroenteritis outbreaks investigated here. They were detected in 14 out of 25 (56%) outbreaks with an identified pathogen. Genotypes producing both sporadic cases and outbreaks were diverse, with a predominance of GGII strains related to genotypes Melksham and Lordsdale. Five strains clustered with a "new variant" designated GGIIb, which was detected circulating throughout quite a few European countries in the years 2000 and 2001. The emergence mechanism of these strains might be the occurrence of intertypic recombinations between different viruses. The nucleotide sequence of part of the capsid gene (ORF2) from three of these strains demonstrated their relationship with Mexico virus.

Antiviral susceptibility of Herpes simplex viruses and its clinical correlates: a single center's experience.

The in vitro susceptibility to acyclovir of 204 herpes simplex virus isolates from 165 immunocompromised patients treated at our hospital was determined by the cytopathic effect reduction assay. Approximately 95% of herpes simplex virus 1 and 73% of herpes simplex virus 2 isolates were inhibited by acyclovir at concentrations of <2 microgram/mL. From 8 patients (5%), an isolate with low susceptibility to acyclovir (50% inhibitory dose, >3 microgram/mL) was recovered. Medical records of 83 patients were reviewed. Lesions resolved in most of the patients, independent of treatment. Treatment failures were not always associated with isolation of an in vitro-resistant virus. On the contrary, when a virus with low susceptibility to acyclovir was isolated, resolution of the lesion was the rule. In 9 of 10 patients with subsequent recurrent episodes of disease, the susceptibility of the viruses isolated was similar to that of the first episode. Routine susceptibility testing in our geographic area is not encouraged because of the low incidence of acyclovir-resistant herpes simplex viruses.

Association between asymptomatic carriage and sporadic (endemic) meningococcal disease in an open community.

We analysed a strain collection representative of the overall Neisseria meningitidis population circulating in an open community (46,000 inhabitants, Spain) during an endemic period (30 isolates from patients and 191 from throat cultures of healthy individuals) by both phenotypic and molecular techniques. Almost all patient isolates were assigned to three hyper-virulent lineages (ET-5 complex, ET-37 complex and cluster A4) by both multilocus enzyme electrophoresis (MEE) and pulsed-field gel electrophoresis (PFGE). In contrast, MEE and PFGE assigned 20% and 15% respectively of carrier isolates to the hyper-virulent clones (4% for both methods together). There was also a higher correlation between PFGE and phenotypes associated with virulent clones. These notable differences between the two molecular methods were further observed in more than half the carrier isolates, suggesting that the associations between these strains were distorted by recombination events. However, almost one-third of total endemic strains from symptom-free carriers and almost all patient strains belonged to clones defined by MEE and PFGE, with no known epidemiological connection. These data indicate low transmission and a weak clonal structure for N. meningitidis.

[In vitro susceptibility to ganciclovir and foscarnet of cytomegaloviruses]. / Sensibilidad in vitro de citomegalovirus al ganciclovir y al foscarnet.

Ganciclovir is the drug of choice for the treatment of acute cytomegalovirus infections. This antiviral agent is a nucleoside analog of guanine whose activity is dependent upon its intracellular phosphorylation to the triphosphate derivative. Foscarnet is used to treat immunosuppressed patients such as organ transplant recipients and AIDS patients with cytomegalovirus who do not tolerate or develop resistance to ganciclovir. Foscarnet is a pyrophosphate analog that directly inhibits viral DNA polymerase. Resistant isolates have been recovered from immunocompromised patients treated with both anticytomegalovirus compounds. The aims of this study were to prepare a plaque reduction assay to study the in vitro susceptibility of cytomegalovirus to ganciclovir and foscarnet, and to apply it to the knowledge of in vitro susceptibility values of cytomegalovirus isolated from clinical samples. Eighty isolates from patients who had never been treated with ganciclovir or foscarnet were tested for antiviral susceptibility. The plaque reduction assay took 6-8 weeks. The results are expressed as ID(50) (inhibitory dose 50), and the ID(50) values of ganciclovir were between 2.14 and 13.49 microM. The ID(50) for ganciclovir was higher that 12 microM in only two cases (2%). The molecular study of the DNA of these did not show any mutation in the UL97 gene. The ID(50) values of foscarnet were between 46.65 and 460.22 microM. In 78 cases (98%) foscarnet ID(50) was lower than 400 microM. These results were comparable with those obtained by other authors. To summarize, the frequency of cytomegalovirus strains resistant in vitro to ganciclovir and foscarnet in previously untreated patients was low and when it was present it did not involve therapeutic failure since the patients progressed favorably.

Dramatic decline of serogroup C meningococcal disease incidence in Catalonia (Spain) 24 months after a mass vaccination programme of children and young people.

STUDY OBJECTIVES: The objective of this study was to evaluate the effectiveness of a mass vaccination programme carried out in Catalonia (Spain) in the last quarter of 1997 in response to an upsurge of serogroup C meningococcal disease (SCMD). DESIGN: Vaccination coverage in the 18 month to 19 years age group was investigated by means of a specific vaccination register. Vaccination effectiveness was calculated using the prospective cohort method. Cases of SCMD were identified on the basis of compulsory reporting and microbiological notification by hospital laboratories. Vaccination histories were investigated in all cases. Unadjusted and age adjusted vaccination effectiveness referred to the time of vaccination and the corresponding 95% confidence intervals (CI) were estimated at 6, 12, 18 and 24 months of follow up. SETTING: All population aged 18 months to 19 years of Catalonia. MAIN RESULTS: A total of seven cases of SCMD were detected at six months of follow up (one in the vaccinated cohort), 12 cases at 12 months (one in the vaccinated cohort), 19 cases at 18 months (two in the vaccinated cohort) and 24 at 24 months (two in the vaccinated cohort). The age adjusted effectiveness was 84% (95%CI 30, 97) at six months, 92% (95%CI 63, 98) at 12 months, 92% (95% CI 71, 98) at 18 months and 94% (95%CI 78, 98) at 24 months. In the target population, cases have been reduced by more than two thirds (68%) two years after the vaccination programme. In the total population the reduction was 43%. CONCLUSION: Vaccination effectiveness has been high in Catalonia, with a dramatic reduction in disease incidence in the vaccinated cohort accompanied by a relevant reduction in the overall population. Given that vaccination coverage was only 54.6%, it may be supposed that this vaccination effectiveness is attributable, in part, to the herd immunity conferred by the vaccine.

[Clinical utility of susceptibility testing of herpes simplex virus to acyclovir]. / Utilidad clínica del estudio de la sensibilidad al aciclovir de los virus herpes simple.

In vitro susceptibility to acyclovir of 96 strains of herpes simplex virus isolated from 80 immunocompromised patients attended in our hospital was studied by the cytopathic effect reduction assay. Ninety-eight percent (61/62) of herpes simplex virus 1 strains and 91% (31/34) of herpes simplex virus 2 strains were inhibited by acyclovir concentrations lower than 3 mg/l. In 5% of the patients herpes simplex strains resistant to acyclovir (ID(50) >3 mg/l) were isolated. Ninety-eight percent of the lesions caused by herpes simplex viruses susceptible to acyclovir (ID(50) <3 mg/l) resolved independently of treatment. In two cases, the cytopathic effect reduction assay was not able to predict treatment failure and persistance of the lesions was not always associated with isolation of a resistant strain in vitro. In four cases, isolation of a strain resistant to acyclovir was not indicative of treatment failure. In conclusion, we believe there is no need to routinely test susceptibility of herpes simplex viruses to acyclovir and that susceptibility testing should be indicated only in patients in whom lesions persist and other causes have been ruled out.

Cloning and sequence of the gene encoding a novel cefotaxime-hydrolyzing beta-lactamase (CTX-M-9) from Escherichia coli in Spain.

A new CTX-M-type beta-lactamase (CTX-M-9) has been cloned from a clinical cefotaxime-resistant Escherichia coli strain. Despite the close identity that exists between the CTX-M-9 and Toho-2 beta-lactamases (88%), the 35 amino acids located between residues Ala-185 and Ala-219 are totally different in both enzymes. Outside of this region there are only six amino acids substitutions between both proteins.

Comparison of IS1245 restriction fragment length polymorphism and pulsed-field gel electrophoresis for typing clinical isolates of Mycobacterium avium subsp avium.

SETTING: Little is still known about the epidemiology and pathogenesis of Mycobacterium avium subsp avium (MASA) infection. OBJECTIVE: Examination of the reproducibility and the stability over time of pulsed-field gel electrophoresis (PFGE) and IS1245 restriction fragment length polymorphism (IS1245-RFLP) techniques. The ability of these typing systems for differentiating clinical isolates of MASA was also assessed. DESIGN: Clinical isolates recovered from 63 patients (59 human immunodeficiency virus [HIV] positive and four HIV-negative) were studied by insertion sequence IS1245 and PFGE. For the study of in vivo and in vitro stability, strains collected over time from four patients and five strains chosen at random, respectively, were used. RESULTS: The stability of PFGE and IS1245-RFLP in vitro was excellent. PFGE was also stable in vivo, but IS1245-RFLP patterns showed some variation. The discriminatory power of IS1245-RFLP and PFGE was 0.995 and 0.989, respectively. The cluster analysis did not reveal differences between strains recovered from HIV-negative and HIV-positive patients or between patients with colonisation, local infection or disseminated disease. CONCLUSION: IS1245-RFLP and PFGE are useful tools for typing MASA strains. However, IS1245 variations in vivo may complicate the analysis of epidemiological relationships.

Antibiotic resistance trends in enteropathogenic bacteria isolated in 1985-1987 and 1995-1998 in Barcelona.

Trends in resistance to antimicrobial agents used for therapy have been evaluated with 3,797 enteropathogenic bacteria, Campylobacter, Salmonella, Shigella, and Yersinia, between 1985-1987 and 1995-1998. The greater increase in the rate of resistance was observed in Campylobacter jejuni for quinolones (from 1 to 82%) and tetracycline (from 23 to 72%) and in gastroenteric salmonellae for ampicillin (from 8 to 44%), chloramphenicol (from 1.7 to 26%), and trimethoprim-sulfamethoxazole and nalidixic acid (from less than 0.5 to 11%). Multidrug resistance was detected in several Salmonella serotypes. In the 1995-1998 period, 76% of Shigella strains were resistant to trimethoprim-sulfamethoxazole, 43% were resistant to ampicillin, and 39% were resistant to chloramphenicol. Seventy-two percent of Yersinia enterocolitica O3 strains were resistant to streptomycin, 45% were resistant to sulfonamides, 28% were resistant to trimethoprim-sulfamethoxazole, and 20% were resistant to chloramphenicol.

Predictors of tuberculosis transmission in prisons: an analysis using conventional and molecular methods.

OBJECTIVE: To determine the tuberculosis (TB) transmission patterns within the prison system in Catalonia, conventional epidemiological techniques were combined with DNA fingerprinting of Mycobacterium tuberculosis. METHODS: IS6110- and polymorphic GC-rich repeat sequence (PGRS)-based restriction fragment length polymorphism (RFLP) were combined with epidemiological studies to assess the relatedness of isolates from all patients with confirmed TB at five prisons in the province of Barcelona (Catalonia, Spain), between 1 July 1994 and 31 December 1996. Risk factors for transmission were analysed to a logistic regression. The extent of drug-resistant TB was also assessed. RESULTS: The incidence of TB during the study period was 2775 cases per 100,000 inmate years. Of the 247 culture-positive cases, 126 (51%) appeared to have active TB as a result of recent transmission. Using conventional epidemiological methods, 14 active chains of transmission were identified in prison involving 65 isolates (52% of clustered patients). A lengthy history of imprisonment [odds ratio (OR) 2.8, 95% confidence interval (CI) 1.52-5.11] and pulmonary TB (OR 2.36, 95% CI 1.17-4.75) were independently associated with clustering. Low rates of both initial (2.9%) and acquired drug resistance (5.8%) were identified and there was no evidence of the transmission of drug-resistant TB. CONCLUSION: In the prison system studied, the recent transmission of TB contributes substantially to the overall incidence of the disease. Both lengthy incarcerations and delays in identifying inmates with pulmonary symptoms play a key role in this recent transmission. Directly observed therapy (DOT) is a critical control strategy for reducing the emergence of drug resistance and for avoiding the transmission of resistant organisms.