Anastomotic leak in colorectal cancer surgery: Contribution of gut microbiota and prediction approaches.

AIM: To monitor prospectively the occurrence of colorectal anastomotic leakage (CAL) in patients with colon cancer undergoing resectional surgery, characterizing the microbiota in both faeces and mucosal biopsies of anastomosis. In a second stage, we investigated the ability to predict CAL using machine learning models based on clinical data and microbiota composition. METHOD: A total of 111 patients were included, from whom a faecal sample was obtained, as well as biopsy samples from proximal and distal sites in the healthy margins of the tumour piece. The microorganisms present in the samples were investigated using microbial culture and 16S rDNA massive sequencing. Collagenase and protease production was determined, as well as the presence of genes responsible for expressing enzymes with these activities. Machine learning analyses were developed using clinical and microbiological data. RESULTS: The incidence of CAL was 9.0%, and CAL was associated with collagenase/protease-producing Enterococcus. Significant differences were found in the microbiota composition of proximal and distal biopsy samples, but not in faecal samples, among patients who developed CAL. Clinical predictors of CAL were 5-day C-reactive protein and heart disease, whereas 3-day C-reactive protein and diabetes were negative predictors. CONCLUSION: Biopsy samples from surgical margins, rather than faecal samples, are the most appropriate samples for exploring the contribution of the intestinal microbiota to CAL. Enterococci are only enriched in the anastomosis after surgery, and their collagenases and proteases are involved in the degradation of the anastomotic scar.

Cefiderocol resistance genomics in sequential chronic Pseudomonas aeruginosa isolates from cystic fibrosis patients.

OBJECTIVE: To evaluate the activity of cefiderocol against sequential P. aeruginosa isolates from chronically-infected cystic fibrosis patients as well as to investigate the potential mechanisms involved in resistance through whole genome sequencing. METHODS: Three sequential P. aeruginosa isolates from each of 50 chronically-colonized cystic fibrosis patients were studied. MICs for novel and classical antipseudomonal agents were determined by broth microdilution and whole genome sequences (n = 150) were obtained to investigate the presence of mutations within a set of chromosomal genes involved in P. aeruginosa antibiotic resistance (n = 40) and iron uptake (n = 120). RESULTS: Cefiderocol showed the lowest MIC50/90 values and its susceptibility rate was comparable to other novel antipseudomonal agents. Clinical resistance was documented in 9 isolates from 6 patients. Resistance genes associated with a statistically significant increase in cefiderocol MICs included ampC, pmrAB, galU, fusA1 and those coding the penicillin-binding proteins PBP2 and PBP3. Likewise, mutations within several genes participating in different iron-uptake systems were found to be significantly associated with resistance, including genes participating in the pyochelin and pyoverdin biosynthesis and several tonB-dependent receptors. Mutator and small colony variants isolates were also associated with increased cefiderocol MICs. DISCUSSION: Cefiderocol resistance is modulated by a complex mutational resistome, potentially conferring cross-resistance to novel beta-lactam beta-lactamase combinations, as well as an extended list of mutated iron-uptake genes. Monitoring the acquisition of mutations in all these genes will be helpful to guide treatments and mitigate the emergence and spread of resistance to this novel antibiotic.

Impacto de un programa de vigilancia activa y medidas de control de infecciones sobre la incidencia de bacilos gram negativos resistentes a carbapenems en una unidad de cuidados intensivos

Resumen Las infecciones hospitalarias causadas por bacilos gram negativos resistentes a carbapenems (BGNCR) están asociadas al aumento de morbimortalidad y gasto sanitario. La identificación mediante cultivos de vigilancia y las medidas de control de infecciones permiten reducir su diseminación. El objetivo del estudio fue evaluar el impacto de un programa de vigilancia integrado a protocolos de control de infecciones sobre la incidencia de BGNCR y conocer su epidemiología molecular en una unidad de cuidados intensivos. Se realizaron auditorías seguidas de un programa de cultivo de vigilancia activa y caracterización molecular de BGNCR, antes y después de la implementación de programas de prevención y control de infecciones. El screening microbiológico se realizó en medios cromogénicos; la caracterización molecular de p-lactamasas (blaKPC, bla0XA-48-like, blaVIM, blaiMP, blaNDM, blaSHV y blaCTx-M) por PCR y la tipificación molecular por PFGE y MLST para Klebsiella pneumoniae. El protocolo desarrollado permitió reducir la colonización global de 16,92% al 9,67%. La diseminación de K. pneumoniae fue a expensas de diversos clones portadores de KPC-2 asociada a BLEE SHV-2 y CTX-M-15, y distribuidos en varios secuenciotipos (ST17, ST13, ST2256, ST353); no se observó persistencia de un clon particular y ningún aislamiento presentó factores de virulencia asociados a hipervi-rulencia. Los aislamientos de Acinetobacter baumannii fueron mayoritariamente productores de IMP-1. El análisis PFGE individualizó 3 clusters, asumiendo que la diseminación fue clonal.

Abstract Hospital-acquired infections caused by carbapenem-resistant Gram-negative bacteria (CRGNB) have been increasingly reported worldwide and are associated with high rates of mortality especially in intensive care units(ICUs). Early identification through rectal surveillance cultures and implementation of infection control measures(ICM) including contact precautions, staff education on cleaning and hand hygiene may reduce the spread of these microorganisms. The aim of this work was to assess the impact of enhanced ICM on CRGNB colonization and to describe the molecular epidemiology of these bacteria in a polyvalent ICU in a tertiary level hospital. A prospective study including audits and active surveillance culture program, with molecular characterization, was conducted before and after the implementation of prevention programs and infection control measures. Microbiological screening was performed in chromogenic media; PCR targeting p-lactamases genes (ó/qkpc, óíQndm, blaviM and blaoxA-48, blasHv and ó/qctx-m), molecular typing by PFGE; and MLST in K. pneumoniae were performed. CRGNB colonization was reduced from 16.92% to 9.67% upon implementing the infection control measures. In K. pneumoniae the most frequent carbapenemase type was KPC-2 associated with SHV-2 and CTX-M-15, and was disseminated in various STs (ST17, ST13, ST2256, ST353); there was no persistence of particular clones and virulence factors showed no association with hypervirulence. IMP-1 carbapenemase predominated in A. baumannii and the PFGE analysis individualized 3 clusters, assuming that the dissemination in the ICU was clonal. The early detection of patients colo-nized with CRBGN by using epidemiological surveillance cultures and the implementation of prophylactic measures are key to reducing the incidence of these microorganisms.

Description of microbial diversity associated with ticks Hyalomma dromedarii (Acari: Ixodidae) isolated from camels in Hail region (Saudi Arabia) using massive sequencing of 16S rDNA.

Ticks are blood feeder able to transmit a wide diversity of microbes including pathogens. Therefore, it is of our interest to detect the diversity of microorganisms residing within ticks using massive sequencing of 16S rDNA. In this study, 200 adult ticks were collected from healthy camels in two localities from Hail province (Saudi Arabia). The analysis showed high microbial diversity dominated by the two domains (Archaea and Bacteria) associated with Hyalomma dromedarii from both regions. Proteobacteria (61.3%) and Firmicutes (31.2%) dominated the ticks from the Al Khotha region. While, the microbiome of ticks from the Al Gayed region was dominated by Proteobacteria (81.2%) and Firmicutes (9.2%). Twenty-three families were identified in the DNA-pool from the Al Gayed region, and was dominated by Pseudomonadaceae (45.37%), and Marinobacteraceae (14.39%) families. Francisellaceae (46%), Staphylococcaceae (24.26%) dominated the microbiome of the ticks collected from Al Gayed region. Thus, the genera Pseudomonas, Francisella, Proteus, Marinobacter, Glutamicibacter, Pedobacter, and Staphylococcus are largely distributed in the two identified microbiomes. This study concluded that ticks collected from the studied localities contained a wide range of microbial communities. These data have a great veterinary and medical importance in near future.

Antimicrobial-resistant Escherichia coli and Enterococcus spp. isolated from Miranda donkey (Equus asinus): an old problem from a new source with a different approach.

PURPOSE: The Miranda donkey (Equus asinus) is an endangeredasinine from Miranda do Douro region, located in the north east of Portugal. We studied the antimicrobial resistance and virulence genes in Escherichia coli and Enterococcus spp. isolates from these animals. METHODOLOGY: In March 2014, a total of 66 faecal samples were recovered from independent animals. Antibiotic resistance was determined by the disc diffusion method. Carriage of genes coding for antibiotic-resistant and virulent factors was analysed by PCR. RESULTS: A total of 66 E. coli and 41 enterococcal isolates were detected, with Enterococcus faecium (61 %) and Enterococcus hirae (24 %) being the most prevalent species. For enterococcal isolates, high percentages of resistance rates to tetracycline (68.3 %), quinupristin/dalfopristin (51.2 %) and ciprofloxacin (48.8 %) were observed. The genes erm(A) and/or erm(B), tet(M) and/or tet(L), vat(D) and/or vat(E) and aph(3')-IIIa were also found. The most frequent virulence gene detected was gel(E), followed by ace, cpd and hyl. Escherichia coli isolates were highly resistant to streptomycin (78 %), whereas 39 % of them exhibited resistance to aminoglycosides and tetracycline. Genes sul1 and/or sul2 were detected in 66.7 % of trimethoprim/sulfamethoxazole-resistant isolates. The virulence genes detected were fim(A) (46 %) and cnf1 (27 %). CONCLUSION: To the best of our knowledge, this is the first report showing antibiotic resistance among Escherichiacoli and Enterococcus spp. isolates from the Miranda donkey in Portugal, indicating possible antibiotic-resistant bacterial reservoirs. However, the detection of these resistances presents a low risk for other animals and human beings in that rural area.

Sharing of bacterial strains between breast milk and infant feces.

In previous years, it has been shown that human milk is a potential source of bacteria for the infant gut. The results of this work confirm the presence of the same specific bacterial strains of Bifidobacterium, Lactobacillus, and Staphylococcus in breast milk and infant fecal samples. The identity of bacteria isolated from breast milk and infant feces from 20 mother-infant pairs was investigated at the strain level. DNA from Staphylococcus, Lactobacillus, and Bifidobacterium was detected by qRTi-PCR in nearly all samples analyzed. These samples were cultured on different agar media. One colony representative of each morphology was selected and identified at the species level combining classical tests and molecular techniques (PCR, RAPD, PFGE, and/or MLST genotyping). Breast milk and infant feces from 19 mother-infant pairs shared different Staphylococcus, Lactobacillus, and/or Bifidobacterium species and strains. Significantly, 2 mother-infant pairs shared 4 bacterial strains although most pairs shared 2. These results confirm that breast milk and infant feces from mother-infant pairs share the same strain(s), indicating that breastfeeding could contribute to the bacterial transfer from the mother to the infant and, therefore, to the infant gut colonization.

Reidentification of Streptococcus bovis isolates causing bacteremia according to the new taxonomy criteria: still an issue?

All Streptococcus bovis blood culture isolates recovered from January 2003 to January 2010 (n = 52) at the Hospital Universitario Ramón y Cajal were reidentified on the basis of their genetic traits using new taxonomic criteria. Initial identification was performed by the semiautomatic Wider system (Fco. Soria-Melguizo, Spain) and the API 20 Strep system (bioMérieux, France). All isolates were reidentified by PCR amplification and sequencing of both the 16S rRNA and sodA genes and by mass spectrometry using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS; Bruker, Germany). Results of 16S rRNA/sodA gene sequencing were as follows: Streptococcus gallolyticus subsp. gallolyticus, 14/14 (number of isolates identified by 16S rRNA/number of isolates identified by sodA gene sequencing); Streptococcus gallolyticus subsp. pasteurianus, 24/24; Streptococcus spp., 7/0; Streptococcus infantarius subsp. infantarius, 0/2; Streptococcus lutetiensis, 0/5; Leuconostoc mesenteroides, 4/0; and Lactococcus lactis, 3/3. MALDI-TOF MS identified 27 S. gallolyticus isolates but not at the subspecies level, 4 L. mesenteroides isolates, 3 L. lactis isolates, and 6 S. lutetiensis isolates, whereas 12 isolates rendered a nonreliable identification result. Pulsed-field gel electrophoresis grouped all S. gallolyticus subsp. gallolyticus isolates into 3 major clusters clearly different from those of the S. gallolyticus subsp. pasteurianus isolates, which, in turn, exhibited no clonal relationship. The percentages of resistance to the tested antimicrobials were 38% for erythromycin, 23% for fosfomycin, 10% for levofloxacin, 6% for tetracycline, and 4% for co-trimoxazole. The most frequent underlying diseases were hepatobiliary disorders (53%), endocarditis (17%), and malignancies (12%). We conclude that sequencing of the sodA gene was the most discriminatory method and that S. gallolyticus subsp. pasteurianus appears to have a higher genetic diversity than S. gallolyticus subsp. gallolyticus.

[Antimicrobial susceptibility and genetic bases for resistance of infection-causing Enterococcus strains in Cuba]. / Susceptibilidad antimicrobiana y bases genéticas de la resistencia de cepas de Enterococcus causantes de infecciones en Cuba.

OBJECTIVE: To identify infection-causing Enterococcus species in Cuban hospitals and determine their susceptibility to antimicrobial drugs, as well as their resistance mechanisms. METHODS: A total of 687 Enterococcus isolates from 30 Cuban hospitals in nine provinces of the country were studied over the period 2000-2009. The species were identified using both the conventional method and the automatic API(®) system. The minimum inhibitory concentration was determined for 13 antimicrobial drugs following the standards recommended by the Clinical Laboratory and Standards Institute. The polymerase chain reaction technique was used to characterize the genes that were resistant to aminoglycosides, erythromycin, tetracycline, and glucopeptides. The presence of beta-lactamase was determined by the chromogenic cephalosporin test. RESULTS: The most prevalent species were Enterococcus faecalis (82.9%) and E. faecium (12.2%). Resistance to glucopeptides (1.0%) was mediated by the vanA and vanB genes. The strains resistant to ampicillin (6%) did not produce beta-lactamases. A high percentage of resistance to aminoglycosides was observed. Gentamicin (31.0%) and streptomycin and amikacin (29.1%) were mediated by the aac(6')Ie-aph(2")Ia, aph(3')-IIIa, ant(6)Ia, and ant(3")(9) genes. A correlation was found between resistance to tetracycline (56.0%) and presence of the tet(M) (75.1%) and tet(L) genes (7.0%), while resistance to erythromycin (34.1%) was due to the erm(B) gene (70.9%). CONCLUSIONS: Resistance to vancomycin is infrequent in Cuba, as opposed to a high level of resistance to aminoglycosides, which may be indicative of treatment failures. The microbiology laboratory is a cornerstone of Enterococcus infection surveillance, along with ongoing monitoring of the susceptibility of these infections to antimicrobial drugs at a time when resistance of this microorganism is on the rise.