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.

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.

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.

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.

Design of clone-specific probes from genome sequences for rapid PCR-typing of outbreak pathogens.

The genome sequence of one OXA-48-producing Klebsiella pneumoniae belonging to sequence type (ST) 405, and three belonging to ST11, were used to design and test ST-specific PCR assays for typing OXA-48-producing K. pneumoniae. The approach proved to be useful for in-house development of rapid PCR typing assays for local outbreak surveillance.

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.

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.

Molecular analysis of community-acquired methicillin-susceptible and resistant Staphylococcus aureus isolates recovered from bacteraemic and osteomyelitis infections in children from Tunisia.

Thirty-six children (27 boys, nine girls) that fulfilled CDC criteria for community-acquired infections were diagnosed with bacteraemia and/or osteomyelitis caused by Staphylococcus aureus during an 18-month period (2006-2008). Antibiotic susceptibility was determined by an agar dilution method. SCCmec type, carriage of pvl genes, agr type and spa-typing were determined using specific PCR protocols. Clonal relatedness was examined by pulsed field gel electrophoresis-SmaI and mutilocus sequence typing techniques. From the 36 isolates, eight (22%) corresponded to methicillin-resistant Staphylococcus aureus (MRSA) -t044/042-CC80/CC5-IVc-pvl(+) -agrIII/II. The highest genetic diversity was observed among the 28 community-acquired methicillin-susceptible S. aureus (CA-MSSA) isolates: 22 spa-variants that also grouped by multilocus sequence typing in CC1, CC5, CC6, CC8, CC30, CC80, CC97 and the singletons ST464, ST1467, ST1468 and ST1469. The pvl genes were detected in all eight CA-MRSA isolates and in eight CA-MSSA isolates (28%), being significantly more frequent among isolates causing osteoarticular infection (11 of 12, 92%) than in the bacteraemic isolates (six of 24, 25%). Based on patients' age, three groups were considered: newborns, infants and children. Bacteraemia was diagnosed in all newborns and infants, whereas in 42% of the children group osteomyelitis was the unique presentation. In most cases, the portal of entry was either the skin or unknown. In general, favourable outcome was observed, except in four cases-three of whom had severe complications and one died. In summary, we analysed the epidemiological and genetic background of community-acquired staphylococcal strains causing bacteraemic and/or osteomyelitis infections in children from Tunisia, describing three new sequence types and one novel spa type.

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.

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.

Changes in P-glycoprotein expression in gastric carcinoma with respect to distant gastric mucosa may be influenced by p53.

BACKGROUND: The effectiveness of some chemotherapeutic agents used to treat gastric carcinoma patients may be impaired by the presence of P-glycoprotein (P-gp) and the status of p53. A modulation of P-gp expression by p53 or other alterations during tumorigenesis have been reported. The authors analyzed P-gp expression in relation to p53 and histopathologic features in gastric carcinoma. METHODS: Forty-one resected gastric carcinomas and mucosa distant from the tumor were assessed for P-gp expression by immunohistochemistry with C494 and JSB-1 antibodies. p53 expression was also immunohistochemically assessed by DO7 antibody in tumor samples. P-gp and p53 expression were semiquantitatively analyzed according to the percentage of stained cells. Histologic type, grade, vessel invasion, and stage were also studied. RESULTS: Moderate or high P-gp expression was detected in gastric carcinoma in 29 cases (71%) and in gastric mucosa remote from the tumor in 36 cases (88%). This reduction in P-gp expression was observed in 22% of the carcinomas, all but 1 being p53 immunonegative tumors. Thus, 8 (42%) of the p53 immunonegative carcinomas showed a loss of P-gp expression compared with their distant gastric mucosa. All p53 immunopositive carcinomas coexpressed P-gp. No correlation between P-gp expression and histologic type, grade, vessel invasion, or stage was found. CONCLUSIONS: P-gp expression in gastric carcinomas is frequent and coexpression with p53 is found. The analysis of P-gp expression in carcinomas and distant mucosa show that it is not regulated by p53, but a loss of P-gp detected in some of these carcinomas is mainly associated with a lack of p53 protein accumulation.

Evaluation of the Wider system, a new computer-assisted image-processing device for bacterial identification and susceptibility testing.

The Wider system is a newly developed computer-assisted image-processing device for both bacterial identification and antimicrobial susceptibility testing. It has been adapted to be able to read and interpret commercial MicroScan panels. Two hundred forty-four fresh consecutive clinical isolates (138 isolates of the family Enterobacteriaceae, 25 nonfermentative gram-negative rods [NFGNRs], and 81 gram-positive cocci) were tested. In addition, 100 enterobacterial strains with known beta-lactam resistance mechanisms (22 strains with chromosomal AmpC beta-lactamase, 8 strains with chromosomal class A beta-lactamase, 21 broad-spectrum and IRT beta-lactamase-producing strains, 41 extended-spectrum beta-lactamase-producing strains, and 8 permeability mutants) were tested. API galleries and National Committee for Clinical Laboratory Standards (NCCLS) microdilution methods were used as reference methods. The Wider system correctly identified 97.5% of the clinical isolates at the species level. Overall essential agreement (+/-1 log(2) dilution for 3,719 organism-antimicrobial drug combinations) was 95.6% (isolates of the family Enterobacteriaceae, 96.6%; NFGNRs, 88.0%; gram-positive cocci, 95.6%). The lowest essential agreement was observed with Enterobacteriaceae versus imipenem (84.0%), NFGNR versus piperacillin (88.0%) and cefepime (88.0%), and gram-positive isolates versus penicillin (80.4%). The category error rate (NCCLS criteria) was 4.2% (2.0% very major errors, 0.6% major errors, and 1. 5% minor errors). Essential agreement and interpretive error rates for eight beta-lactam antibiotics against isolates of the family Enterobacteriaceae with known beta-lactam resistance mechanisms were 94.8 and 5.4%, respectively. Interestingly, the very major error rate was only 0.8%. Minor errors (3.6%) were mainly observed with amoxicillin-clavulanate and cefepime against extended-spectrum beta-lactamase-producing isolates. The Wider system is a new reliable tool which applies the image-processing technology to the reading of commercial trays for both bacterial identification and susceptibility testing.

Comparative study of two androgen-induced markers (apolipoprotein D and pepsinogen C) in female and male breast carcinoma.

BACKGROUND: Male breast cancer (MBC) is a rare tumor in comparison to the same disease in the female population (FBC). Classical prognostic factors (tumor size, node status, estrogen receptor positivity, histological grade) have a similar prognostic value in both tumors, but MBC seems to have a worse outcome. Several markers under estrogen control have been shown with a similar incidence in breast tumors of both sexes, while androgen-induced markers have been detected in a higher percentage of breast tumors in males. AIMS AND METHODS: Our purpose was to compare in 68 MBC and in 68 FBC the expression of Pepsinogen C (Pep C) and Apolipoprotein D (Apo D), two proteins under androgen control, by immunohistochemical methods. RESULTS: Pep C was expressed by 52 of 68 (76.4%) MBC patients, whereas 34 of 68 (50%) FBC showed positive staining. Apo D was expressed by 57 of 68 (83.8%) MBC patients, while 40 of 68 (41.2%) FBC stained positively. Differences between percentages of positive expression were significant (p<0.005 for Pep C; p<0.0001 for Apo D). Moreover, differences between expression levels of Pep C and Apo D in both populations of patients were significant. The mean value of Pep C was significantly higher in male breast tumors (HSCORE = 141.3) than in the females (HSCORE = 80.3) (p<0.0001). Similarly, Apo D mean value was significantly higher in MBC (HSCORE = 161.5) than in FBC (HSCORE = 102.3) (p=0.006). CONCLUSION: These differences can open the field of a more selective hormonal therapy that should not be based on estrogen receptor status only, but also on androgen receptor status.