Genomic Surveillance Uncovers a 10-Year Persistence of an OXA-24/40 Acinetobacter baumannii Clone in a Tertiary Hospital in Northern Spain.

Infections caused by carbapenem-resistant Acinetobacter baumannii are a global threat causing a high number of fatal infections. This microorganism can also easily acquire antibiotic resistance determinants, making the treatment of infections a big challenge, and has the ability to persist in the hospital environment under a wide range of conditions. The objective of this work was to study the molecular epidemiology and genetic characteristics of two blaOXA24/40Acinetobacter baumannii outbreaks (2009 and 2020-21) at a tertiary hospital in Northern Spain. Thirty-six isolates were investigated and genotypically screened by Whole Genome Sequencing to analyse the resistome and virulome. Isolates were resistant to carbapenems, aminoglycosides and fluoroquinolones. Multi-Locus Sequence Typing analysis identified that Outbreak 1 was mainly produced by isolates belonging to ST3Pas/ST106Oxf (IC3) containing blaOXA24/40, blaOXA71 and blaADC119. Outbreak 2 isolates were exclusively ST2Pas/ST801Oxf (IC2) blaOXA24/40, blaOXA66 and blaADC30, the same genotype seen in two isolates from 2009. Virulome analysis showed that IC2 isolates contained genes for capsular polysaccharide KL32 and lipooligosacharide OCL5. A 8.9 Kb plasmid encoding the blaOXA24/40 gene was common in all isolates. The persistance over time of a virulent IC2 clone highlights the need of active surveillance to control its spread.

Mercury-induced aggregation of human lens γ-crystallins reveals a potential role in cataract disease.

Cataract disease results from non-amyloid aggregation of eye lens proteins and is the leading cause of blindness in the world. A variety of studies have implicated both essential and xenobiotic metals as potential etiological agents in cataract disease. Essential metal ions, such as copper and zinc, are known to induce the aggregation in vitro of human γD crystallin, one of the more abundant γ-crystallins in the core of the lens. In this study, we expand the investigation of metal-crystallin interactions to heavy metal ions, such as divalent lead, cadmium and mercury. The impact of these metal ions in the non-amyloid aggregation, protein folding and thermal stability of three homologous human lens γ-crystallins has been evaluated using turbidity assays, electron microscopy, electronic absorption and circular dichroism spectroscopies. Our results show that Hg(II) ions can induce the non-amyloid aggregation of human γC and γS crystallins, but not γD crystallin. The mechanism of Hg-induced aggregation involves direct metal-protein interactions, loss of thermal stability, partial unfolding of the N-terminal domain of these proteins, and formation of disulfide-bridged dimers. Putative Hg(II) binding sites in γ-crystallins involved in metal-induced aggregation are discussed. This study reveals that mercury ions can induce the aggregation of human lens proteins, uncovering a potential role of this heavy metal ion in the bioinorganic chemistry of cataract disease.

Photodynamic therapy for the treatment of complex anal fistula.

BACKGROUND: Photodynamic therapy (PDT) is a new procedure for the treatment of anal fistula. This preliminary study was designed to investigate the safety and effectiveness of this new technique in the treatment of anal fistula. METHODS: Ten patients were treated with PDT. Intralesional 5-aminolevulinic acid (ALA) 2% was directly injected into the fistula. The internal and external orifices were closed. After an incubation period of 2 h, the fistula was irradiated using an optical fibre connected to a red laser (MULTIDIODE 630 PDT, INTERmedic, Spain) operating at 1 W/cm for 3 min (180 Joules). Patient demographics, operation notes and complications were recorded. RESULTS: There were no complications. The average length of patient follow-up was 14.9 months (range 12-20 months). We could observe primary healing in eight patients (80%). Two patients (20%) showed persistence of suppuration after the operation. No patient reported incontinence postoperatively. CONCLUSIONS: PDT is a potential sphincter-saving procedure that is safe, simple and minimally invasive and has a high success rate.

Evaluation of the EUCAST disc diffusion susceptibility testing method for Haemophilus influenzae based on the resistance mechanism to ß-lactam antibiotics.

OBJECTIVES: EUCAST developed an antibiotic susceptibility testing method for Haemophilus influenzae. We assessed the EUCAST testing method and EUCAST clinical breakpoints and newly proposed epidemiological cut-off values against H. influenzae clinical isolates with known molecular mechanisms of resistance to ß-lactam antibiotics. METHODS: In total, 89 clinical isolates were used: 30 were ß-lactamase negative with PBP3 mutations (gBLNAR), 20 were ß-lactamase positive without PBP3 mutations (gBLPAR), 15 were ß-lactamase positive with PBP3 mutations (gBLPACR), and 24 were ß-lactamase negative without resistance mechanism (gBLNAS). Twelve different ß-lactam antibiotics and disc charges were tested. RESULTS: None of the discs tested fully separated between gBLNAS and gBLNAR populations. According to EUCAST clinical zone diameter breakpoints, overall the best values of sensitivity and specificity were obtained with cefuroxime 30 µg and amoxicillin/clavulanic acid 2/1 µg discs for detection of gBLNAR and gBLPACR populations, although a previous ß-lactamase test was needed. Other antibiotic discs could be suitable for epidemiological purposes, such us penicillin 10 U for separating gBLNAR isolates and cefoxitin 30 µg for detection of gBLPACR isolates. By Etest using the EUCAST method, the EUCAST MIC clinical breakpoints for ampicillin and amoxicillin/clavulanic acid showed high specificity, but low sensitivity, for the detection of genotypes with mutations in PBP3. CONCLUSIONS: The main genotypes of ß-lactam-resistant H. influenzae can be separated by using the EUCAST disc diffusion method, although it should be noted that overlapping between populations with and without PBP3 mutations is common.

Novel mechanisms of resistance to ß-lactam antibiotics in Haemophilus parainfluenzae: ß-lactamase-negative ampicillin resistance and inhibitor-resistant TEM ß-lactamases.

OBJECTIVES: To determine the mechanisms of resistance to ß-lactam antibiotics in clinical isolates of Haemophilus parainfluenzae. METHODS: Twenty clinical isolates of H. parainfluenzae with decreased susceptibility to aminopenicillins were examined and compared with a control group of 20 fully susceptible isolates. In this collection, the presence of amino acid substitutions in the transpeptidase domain of penicillin-binding protein 3 (PBP3), ß-lactamase production and the surrounding genetic regions of blaTEM genes in selected isolates were analysed. RESULTS: Of the 20 non-susceptible isolates, 8 produced TEM ß-lactamase (gBLPAR), 7 had mutations in the transpeptidase domain of the ftsI gene related to decreased susceptibility to ß-lactams (gBLNAR) and 5 had both resistance mechanisms (gBLPACR). No resistance mechanisms were identified in the susceptible control group (gBLNAS). gBLNAR isolates had MIC90 values 4- to 16-fold higher than gBLNAS isolates for ampicillin, amoxicillin/clavulanic acid, cefuroxime, cefotaxime and cefixime, and the most common PBP3 mutation was Asn526Ser. The additional Ser385Thr substitution (III-like group) may confer decreased susceptibility to cefotaxime, cefixime and aztreonam, as in Haemophilus influenzae. In two ß-lactamase-positive isolates without PBP3 mutations, the inhibitor-resistant TEM (IRT) ß-lactamases TEM-34 and the novel TEM-182 were detected and carried by a TnA transposon of the Tn2 type; both isolates had an amoxicillin/clavulanic acid MIC of ≥8 mg/L. The TnA transposons of two ß-lactamase-positive isolates (TEM-1 and TEM-182) were inserted between the tfc20 and tfc21 genes, typically associated with integrative and conjugative elements in Haemophilus spp.; the TEM-34 IRT ß-lactamase was harboured in a ∼5.5 kb plasmid. CONCLUSIONS: Clinical isolates of H. parainfluenzae express a variety of aminopenicillin resistance mechanisms, either alone or in combination, including PBP3 modifications, blaTEM-1 and IRT ß-lactamase production.

In vivo selection of KPC-94 and KPC-95 in Klebsiella pneumoniae isolates from patients treated with ceftazidime/avibactam.

Ceftazidime/avibactam (CZA) is used to treat infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp). Resistance to CZA is commonly related to point mutations in the blaKPC gene. Here we describe the in vivo emergence of CZA resistance in clinical isolates of KPC-Kp from four patients treated with this combination therapy. Four pre-therapy and five post-therapy KPC-Kp isolates were examined. Antibiogram (microdilution and gradient strips) and whole-genome sequencing were performed. The role of KPC mutations was validated by cloning blaKPC genes into competent Escherichia coli. All KPC-Kp isolates recovered before treatment with CZA were susceptible to CZA and produced KPC-3. Five KPC-Kp isolates recovered after treatment were resistant to this combination. Three post-therapy isolates from two patients produced KPC-31 (D179Y mutation). Additionally, we identified the novel substitution LN169-170H (KPC-94) in one isolate, and the combination of two independently described mutations, D179Y and A172T (KPC-95), in another isolate. All KPC-Kp isolates belonged to sequence type 512 (ST512). All CZA-resistant isolates with blaKPC variants had restoration of carbapenem susceptibility. In conclusion, resistance to CZA was related to blaKPC mutations, including the new KPC-94 and KPC-95 alleles, which do not cause carbapenem resistance.

RedLabRA; a Spanish Network of Microbiology Laboratories for the Surveillance of Antibiotic Resistant Microorganisms.

There is an urgent need to control the clinical and public health impact that antibiotic resistance (AR) causes worldwide. Any measure for its control must be based on an up-to-date and comprehensive knowledge of the situation. However, it is difficult to determine the current dimension of AR because a large part of the available information is based on heterogeneous, insufficiently unified and retrospective data. The integration of genomic information in the surveillance of AR is another important factor for improvement. The Spanish Network of Laboratories for the Surveillance of Resistant Microorganisms (RedLabRA) is a structured network of interconnected microbiology laboratories developed within the Spanish National Plan against Antibiotic Resistance. Its main objective is to support the diagnosis of resistance to antibiotics, integrating molecular characterization in the surveillance.

Self-esteem and suicidal behaviour in youth: A meta-analysis of longitudinal studies.

BACKGROUND: Previous literature suggests that low self-esteem is a risk factor for suicide attempts, but no meta-analyses have been conducted to assess this association in adolescents/young adults. The present study examined the relationship between low self-esteem and suicide attempts in young people (12-26 years old). METHOD: Meta-analyses were performed using random-effects models (ES) and odds ratio (OR). Heterogeneity and sensitivity analyses were performed. RESULTS: From 26,883 initial titles, 22 studies met the inclusion criteria, of which 9 studies had data that could be included in the meta-analysis. The meta-analysis showed that youths with lower self-esteem were more likely to have future suicide attempts, with an effect size (self-esteem as continuous variable) of d = .58 (95% CI = .44 - .73) and, for low self-esteem (categorical variable) an OR = 1.99 (95% CI = 1.39-2.86; p < .001). CONCLUSION: A low level of self-esteem is a risk factor for suicide attempts in adolescents/young adults.

In vivo attenuation and genetic evolution of a ST247-SCCmecI MRSA clone after 13 years of pathogenic bronchopulmonary colonization in a patient with cystic fibrosis: implications of the innate immune response.

Methicillin-resistant Staphylococcus aureus (MRSA) causes chronic pulmonary infections in patients with cystic fibrosis (CF). This study tracks the 13-year evolution (1996-2009) of a single MRSA clone in a male patient with CF, evaluating both the host immunogenic response and the microbial variations. Whole-genome sequencing was performed for the initial (CF-96) and evolved (CF-09) isolates. The immunogenicity of CF-96 and CF-09 was evaluated by incubation with innate immune cells from healthy volunteers. We also studied the patient's innate immune response profile, cytokine production, expression of triggering receptor expressed on myeloid cells-1 (TREM-1), and phagocytosis. A total of 30 MRSA ST247-SCCmecI-pvl(-) isolates were collected, which evidenced a genome size reduction from the CF-96 ancestor to the evolved CF-09 strain. Up to six changes in the spa-type were observed over the course of the 13-year evolution. Cytokine production, TREM-1 expression, and phagocytosis were significantly lower for the healthy volunteer monocytes exposed to CF-09, compared with those exposed to CF-96. Patient monocytes exhibited a reduced inflammatory response when challenged with CF-09. Genetic changes in MRSA, leading to reduced immunogenicity and entry into the refractory state, may contribute to the attenuation of virulence and efficient persistence of the bacteria in the CF lung.

Remission of systematic lupus erythematosus.

The occurrence and characteristics of remissions in patients with systematic lupus erythematosus (SLE) have not been determined. We therefore studied this in a cohort of 667 patients and found that 156 patients had achieved at least 1 period of 1 year or more of treatment-free clinical remission. This represents an incidence density of 0.028 new cases/person/year. Remission occurred within the first 2 years of disease in 62 patients. The mean duration of first remission was 4.6 years (range, 1-21 yr), and 81 patients were still in the initial remission up until cutoff time. Half of the remaining 75 patients who flared after achieving remission have not entered again in remission. Twenty-six of the 38 patients who did remained in remission, and the remaining 12 had subsequent flares and remissions. Treatment-free remission accounted for a mean of 5.8 years, corresponding to half the time of follow-up. Remission was not limited to patients with mild disease: at least 41 patients achieved remission despite renal involvement, 19 had had neuropsychiatric lupus, 15 had had thrombocytopenia, and 8 had had hemolytic anemia. We also found that the longer the time lapse between the initial manifestation and the diagnosis of SLE, the less likely it was for a patient to enter into remission. There was a continuous increase in likelihood of achieving a first remission from the beginning of disease up to 30 years of disease duration, when it reached 70%. Patients who achieved remission had increased survival, independently of the effect of other disease manifestations that cause increased mortality. We conclude that a significant proportion of patients with SLE, including those with severe organ involvement, may become symptom-free and in need of no more medication, perhaps indefinitely. Our findings support the notion that, in general, SLE is a more benign disease than previously considered.

Decrease in serum antiphospholipid antibody levels upon development of nephrotic syndrome in patients with systemic lupus erythematosus: relationship to urinary loss of IgG and other factors.

PURPOSE: Having observed a decrease in antiphospholipid antibodies (aPL) upon the development of nephrotic syndrome, as well as a negative association between nephrotic syndrome and secondary antiphospholipid syndrome, in patients with systemic lupus erythematosus (SLE), we sought to determine if this could be due to urinary loss of aPL and/or other factors. SUBJECTS AND METHODS: IgG and IgM aPL as well as other autoantibodies were studied by enzyme-linked immunosorbent assay with cardiolipin as antigen in serum and urine from six patients with SLE who had elevated serum aPL levels and developed nephrotic syndrome (cases). For controls, we studied: (1) three SLE patients with nephrotic syndrome but low aPL levels; (2) three patients with non-SLE nephrotic syndrome; (3) three SLE patients with high-titer aPL but no proteinuria; and (4) 10 healthy volunteers. RESULTS: We found urinary IgG, but no IgM, aPL in all cases and in one control from Group 2. Serum IgG aPL had gradually decreased after the development of nephrotic syndrome and had become normal. IgM aPL had also decreased in the four patients who had elevated levels, having reached normal levels at the time of the study in two. There was an apparent correlation between serum and urine IgG aPL levels but not between urinary IgG aPL and total proteinuria. By Farr's method, we found no urinary anti-DNA despite high serum titers in three cases. The two cases and one of the controls in Group 1 who had serum antibodies to extractable antigens also had these antibodies in the urine. CONCLUSION: Urinary loss of IgG aPL during nephrotic syndrome does not completely explain the reduction in serum aPL, since IgM also decreases. There could also be decreased synthesis and/or increased catabolism of immunoglobulins.

Preliminary classification criteria for the antiphospholipid syndrome within systemic lupus erythematosus.

Ten percent of 667 consecutive systemic lupus erythematosus (SLE) patients were considered to have definite antiphospholipid syndrome (aPLS) because they had two or more antiphospholipid (aPL)-related clinical manifestations and aPL titers more than 5 SD above the mean of normal controls. Another 14% had either one aPL-related manifestation but high titers of the antibody or two manifestations and low aPL titers (probable aPLS). One fourth of the patients had no manifestations but high titers, one manifestation and low titers, or two or more manifestations and negative aPL titers ("doubtful" aPLS); the other half were considered negative for aPLS. In patients with high-titer aPL, the number of aPL-related manifestations was influenced by disease duration and number of pregnancies, indicating potential mobility of category with time or with risk of recurrent pregnancy loss. Patients with two or more manifestations but variable aPL levels differed in immunosuppressive treatment and in the number of times they had been tested, indicating potential mobility of category with lower treatment and/or further aPL testing. Patients with definite aPLS had increased risk of cutaneous vasculitis, peripheral neuropathy, seizures, psychosis, transient ischemic attacks, and leukopenia. In 11 of 52 SLE patients with definite aPLS the initial manifestation was related to aPL, and in 16 it concurred with an unrelated one. Only two patients fulfilled criteria for aPLS before having other evidence of SLE. The authors conclude that aPLS occurring within SLE is part of the disease rather than an associated condition and propose the use of definite and probable classification categories. These criteria, with appropriate follow-up and clinical and serological exclusion clauses for potential primary conditions, could also be applied to primary aPLS.

Validation of the VITEK2 and the Advance Expert System with a collection of Enterobacteriaceae harboring extended spectrum or inhibitor resistant beta-lactamases.

The susceptibility testing accuracy of the VITEK2 system and the ability of the Advance Expert System (AES) to provide interpretive readings were evaluated against 86 extended spectrum (ESBL) and 6 inhibitor-resistant-TEM (IRT) beta-lactamases producing Enterobacteriaceae clinical isolates. VITEK2 MICs of 12 beta-lactams were compared with those obtained by the standard NCCLS microdilution technique. The overall essential agreement ( +/- 1 log dilution) was 87.8%. Discrepancies were mainly observed with cefepime (30.3% of total number of discrepancies), ceftazidime (21.2%), and cefotaxime (15.1%). MIC discrepancies were slightly higher in CTX-M- (14.4%) than in TEM- (12.5%) or SHV- (11.9%) type ESBL producers and were rare in IRT producers (1.4%). Overall interpretive agreement was 92.5% and minor, major, and very major errors were 5.4%, 1.7%, and 2.1%, respectively. The AES was able to identify an ESBL phenotype in 85 out of 86 isolates (98.8%) and an IRT phenotype in all 6 isolates harboring these enzymes, thus reducing very major errors to 0.9%. The VITEK2 system, in conjunction with the AES software, is a reliable tool for detection of ESBL or IRT producing Enterobacteriaceae isolates.

Study of pancreatic secretion after subtotal gastrectomy followed by different methods of reconstruction.

To study the effect subtotal gastrectomies have on exocrine pancreatic function, 12 dogs were prepared with gastric and duodenal cannulas using a modified technique of Thomas. In our study protocol, we collected pancreatic juice by selective ductal cannulation after having applied two types of stimulation [tryptophan intraduodenally together with secretin intravenously (i.v.) and cholecystokinin i.v. together with secretin i.v.]. Our results did not show a significant increase in pancreatic juice after performing the gastrectomies. In both cases the concentration of bicarbonate decreased significantly in contrast to that in the control group. After Billroth I anastomosis, however, a significant decrease in the first periods of stimulation in relation to the gastrojejunostomy was appreciated. In the case of proteins, a loss of the dose/effect relation in these parameters was seen only after Billroth II anastomosis.

Performance of the VITEK2 system for identification and susceptibility testing of routine Enterobacteriaceae clinical isolates.

The VITEK2 system was evaluated with 138 fresh consecutive routine clinical Enterobacteriaceae isolates. Susceptibility results to 10 beta-lactams, three aminoglycosides and a quinolone were compared with those obtained following the NCCLS standard microdilution. API20E was used as reference method for identification. All but three isolates were correctly identified in 3 h at species level (97.8%), two isolates (1.4%) at genus level and only one isolate was misidentified. Overall essential agreement for susceptibility testing was 97.1%. Discrepancies were mainly observed with piperacillin (1.1%), cefuroxime (0.6%) and amoxycillin/clavulanate (0.3%). Discrepancies for aminoglycosides and ciprofloxacin were low (<0.1%). Minor, major and very major errors (NCCLS categories) were 4.1%, 0.2% and 6.1%, respectively. Very major errors were due to piperacillin (4.5%), ampicillin (0.8%) and amoxycillin/clavulanate (0.8%). The VITEK2 system gave accurate identification and susceptibility testing results of routine Enterobacteriaceae clinical isolates.

Gastric carcinoma: study of the most eminent prognostic factors.

In order to analyse some prognostic factors in relation to gastric cancer, 218 patients were included in a prospective protocol at 'Hospital General de Elche' (Spain) with a follow-up of 100%. Survival curves were calculated using Kaplan-Meier analysis and compared using the log-rank test. The number of patients who underwent resection was 122 (63.5%). In 70 cases (36.5%) the resection was not possible. The postoperative mortality was 4.9%. In the survival of patients with gastric cancer we find the following prognostic factors are significant: age, tumour site, macroscopic type, depth of gastric wall affection, presence of lymph nodes, staging, resectability and surgical intention (curative vs palliative).

[Immuno-nutritional prognostic factors in acute cholecystitis]. / Estudio de factores pronósticos inmunonutricionales en la colecistitis aguda.

A set of parameters that could be used to identify possible immunonutritional disturbances and serve as clinical evolution markers were measured in 210 patients diagnosed of acute cholecystitis (136 females and 74 males, 64.8 and 35.2% respectively) who were admitted into the Surgery Service of the Hospital General de 1++Elche. The prognostic evaluation indices were: a) Nutritional: hematocrit, hemoglobin, total protein, albumin, transferrin; b) Immunological: total lymphocytes and delayed hypersensitivity reaction test. The results obtained were analyzed and compared to those of the literature in this field.

[Metabolic disorders in the gastrectomized patient]. / Estudio de los trastornos metabólicos en el enfermo gastrectomizado.

The present study attempts on one hand to study the metabolic disorders which may present themselves in the gastrectomized patient, such as the malabsorption of fat, vitamin B12, folic acid and iron as well as the possible correlation between steatorrhea and the presence of exocrine pancreatic insufficiency. For this purpose a group of 71 patients have been studied who have undergone a subtotal gastrectomy (70.42%) or total (29.58%) in the General Surgery Services of Elche. The results obtained show the presence of ferropenic or megaloblastic anemia in 61.97% of the group, serious steatorrhea in only 3 patients (4.22%) and calciumphosphorous metabolism alterations appeared in 21.13%. With this we conclude that anemia is the most frequent ferropenic alteration in the gastrectomy patient; steatorrhea does not seem to be produced exclusively by the presence of exocrine pancreatic insufficiency and bone alterations in the gastrectomized patient appear in an insidious manner, being more a question of biochemical alterations than actual clinical lesions.

[An experimental study of the efficacy of politetrafluoroethylene grafts in tracheal surgery]. / Estudio experimental de la eficacia de injertos de politetrafluoroetileno en cirugía traqueal.

We studied the efficacy of polytetrafluoroethylene (PTFE) grafts in the surgical correction of extensive tracheal defects in rabbits. Results showed anastomotic stenosis in 100% of the animals after primary reconstruction when a total of six complete rings were resected. We did not appreciate tracheal stenosis in no animal when we applied a PTFE prosthesis placed around the anastomotic site. Results were not satisfactory upon repairing partial tracheal defect by means of a spindle of PTFE prosthesis. We concluded that the external tutor is useful for the surgical correction of the tracheal stenosis induced after resection of six rings in the cervical tracheal of the animal.