Gut Microbiota Modulation and Prevention of Dysbiosis as an Alternative Approach to Antimicrobial Resistance: A Narrative Review.

Background: The importance of gut microbiota in human health is being increasingly studied. Imbalances in gut microbiota have been associated with infection, inflammation, and obesity. Antibiotic use is the most common and significant cause of major alterations in the composition and function of the gut microbiota and can result in colonization with multidrug-resistant bacteria. Methods: The purpose of this review is to present existing evidence on how microbiota modulation and prevention of gut dysbiosis can serve as tools to combat antimicrobial resistance. Results: While the spread of antibiotic-resistant pathogens requires antibiotics with novel mechanisms of action, the number of newly discovered antimicrobial classes remains very low. For this reason, the application of alternative modalities to combat antimicrobial resistance is necessary. Diet, probiotics/prebiotics, selective oropharyngeal or digestive decontamination, and especially fecal microbiota transplantation (FMT) are under investigation with FMT being the most studied. But, as prevention is better than cure, the implementation of antimicrobial stewardship programs and strict infection control measures along with newly developed chelating agents could also play a crucial role in decreasing colonization with multidrug resistant organisms. Conclusion: New alternative tools to fight antimicrobial resistance via gut microbiota modulation, seem to be effective and should remain the focus of further research and development.

Kinetics and Role of Pancreatic Stone Protein and Midregional Proadrenomedullin as Predictors of Sepsis and Bacteremia in Children with Hematological Malignancies.

Background: To investigate the kinetics and prognostic value of pancreatic stone protein (PSP) and mid-regional proadrenomedullin (MR-proADM) during episodes of febrile neutropenia (FN) in children with hematological malignancies. Material and methods: We evaluated prospectively a total of 70 FN episodes in 70 children with acute leukemias and lymphomas. CRP, PSP, and MR-proADM levels were measured at the onset of the febrile episode (day 1), day 3, and day 7. The outcome and survival of children were evaluated during the study period until day 28. The performance of each marker in identifying sepsis or severe sepsis was assessed as an area under a receiver operating characteristic (ROC) curve. ROC curves were used for each biomarker to derive cut-offs for sensitivity and specificity in distinguishing sepsis from non-sepsis. Results: During the 2-year study period, 70 febrile neutropenia episodes in 70 children with hematological malignancies were enrolled. Of 70 episodes of febrile neutropenia, in 17 (24%), a bacterial/fungal infection was documented. Criteria for sepsis were fulfilled for 31 (44%) and 7 (10%) patients were admitted to PICU. The median values of all biomarkers on day 1 differed significantly between patients with and without sepsis. PSP, MR-proADM, and CRP specificity were 0.82, 0.70, and 0.57, respectively. The sensitivity of PSP, MR-proADM, and CRP were 0.84, 0.74, and 0.88, respectively. Conclusions: PSP and MR-proADM are promising biomarkers for early diagnosis of sepsis during FN episodes in children with hematological malignancies. However, PSP has a higher sensitivity and specificity.

Polyclonal outbreak of vancomycin-resistant Enterococcus faecium in a pediatric oncology department.

We present a polyclonal outbreak of vancomycin-resistant enterococci (VRE) colonization in a pediatric oncology department and the role of a bundle of actions. After the occurrence of VRE bloodstream infections in 2 patients, an active surveillance of VRE colonization was started. Enhanced infection control measures and closure of the department to new admissions for the first 3 months were implemented. Among 32 patients screened for VRE, 21 were found colonized. Daily prevalence of VRE colonization among hospitalized patients ranged from 40% to 75%, but no new VRE infections occurred. Monthly incidence of VRE colonization decreased from 2.5 to 0.6 cases per 100 occupied bed-days at the end of this outbreak by the implementation of the above-mentioned measures. All VRE isolates tested were Enterococcus faecium carrying VanA gene. Pulsed field gel electrophoresis showed a polyclonal outbreak. A case-control study did not show any particular risk factors for colonization. High use of glycopeptide was noted before study outbreak that was drastically decreased during the study but only temporarily. Control of VRE in pediatric oncology departments with high colonization rates is challenging and requires a multifaceted strategy. Polyclonal spread of VRE found in this study suggests a possible effect of prior antimicrobial overuse and the critical need for antimicrobial stewardship especially in the era of multidrug-resistant bacteria.

Life-threatening infection due to community-acquired methicillin-resistant Staphylococcus aureus: case report and review.

We report an unusual case of serious, multifocal, invasive infection due to community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) in a 10-year-old girl with favorable outcome. The child manifested femoral osteomyelitis, pyomyositis, deep femoral vein thrombosis, pneumonia, encephalopathy, and disturbances of almost all organs. She remained in a critical condition for a week. Fever persisted for 6 weeks and acute phase reactants remained increased for 6 months, necessitating a 7-month antistaphylococcal therapy with a glycopeptide and clindamycin. This led to resolution of infection-associated problems during the subsequent 36 months of follow-up. CA-MRSA strain isolated from the patient harbored both staphylococcal chromosomal cassette type IV (SCCmec type IV) and Panton-Valentine leukocidin genes. A literature review of serious CA-MRSA infections indicated that only a small minority of published cases had favorable outcome.

Immune response parameters during labor and early neonatal life.

AIM: Selected cytokines, associated with Th1 and Th2 immune response and inflammation, were studied in order to evaluate the relation between their release into maternal and neonatal circulation, during labour, and after birth, in comparison with those in adults. MATERIALS AND METHODS: Cytokine concentrations were determined by very sensitive immunoassays, in maternal serum (MS), umbilical cord (UC), neonatal serum, the 1st (1N) and 5th (5N) day postpartum and in adult controls. RESULTS: Both IL-2 and IL-4 cytokine concentrations in UC were markedly elevated, compared to adult and MS ones. IL-2 decreased significantly in 5N, while IL-4 remained unchanged. IFN-gamma UC values were significantly lower than those in adults and MS, increasing significantly in 5N. Neonatal serum sIL-2R and sIL-4R were markedly higher than those in adults and MS. IL-1beta, IL-6, sIL-6R, sTNFRI and sTNFRII concentrations in MS and all with TNF-alpha in neonatal serum were significantly higher than in adults. IFN-gamma, IL-1beta, IL-6, TNF-alpha, IL-2R, IL-4R concentrations in MS, 1N and 5N were dependent on the mode of delivery. CONCLUSION: The results of this comparative study are indicative for a meaningful role for the studied cytokines and their receptors in: i) the development of neonatal immune system, ii) the regulation of immune response during labour and early life, and iii) the initiation of the processes of labour.

Molecular epidemiology of outbreak-related pseudomonas aeruginosa strains carrying the novel variant blaVIM-17 metallo-beta-lactamase gene.

A study was designed to investigate the molecular epidemiological characteristics of multidrug-resistant outbreak-related Pseudomonas aeruginosa isolates collected in a university hospital in northern Greece. Of 29 nonreplicate P. aeruginosa isolates resistant to carbapenems and ceftazidime, 14 were positive for metallo-beta-lactamase production. PCR analyses with primers specific for bla(VIM) and bla(IMP) revealed that 13 isolates carried a novel bla(VIM-2) gene variant, designated bla(VIM-17), and only 1 isolate carried bla(VIM-2), a gene predominant among P. aeruginosa strains in Greek hospitals. Pulsed-field gel electrophoresis of XbaI-digested genomic DNAs showed a close genetic relationship for 12 of 13 bla(VIM-17)-carrying outbreak-related isolates, which were of the O11 serotype; the clonally unrelated isolate carrying bla(VIM-17) was of the O12 serotype. PCR mapping strategies for the detection of class 1 integrons and sequencing approaches revealed the presence of integrons containing one bla(VIM) cassette flanked by two aacA29 cassettes. These integrons were similar but not identical to In59 (GenBank accession number AF263519) initially described in France. All isolates carrying bla(VIM-17), regardless of their genetic profile, had an identical integron, named In59.3, indicating that although the hospital outbreak was mainly due to clonal dissemination, the horizontal transmission of the bla(VIM-17)-containing integron among P. aeruginosa isolates should also have occurred. An outbreak-related isolate and a control strain, both of which carried the bla(VIM-2) gene but which were clonally distinct, had an identical integron, named In59.2, which differed only at the level of the bla(VIM) gene from In59.3 integrons, suggesting a common ancestry. The spread of the bla(VIM-17)-containing integron in clonally unrelated P. aeruginosa isolates without any evidence of plasmid carriage is probably associated with a transposon.

Clonal spread of KPC-2 carbapenemase-producing Klebsiella pneumoniae strains in Greece.

OBJECTIVES: KPC-possessing Klebsiella pneumoniae have been found to be widespread in several regions but are still rarely detected in Europe. We describe the characteristics of an outbreak caused by KPC producers in a tertiary care Greek hospital. METHODS: During a 12 month period (October 2007-September 2008), 47 patients in Hippokration University Hospital yielded K. pneumoniae isolates that exhibited reduced susceptibility to carbapenems and were phenotypically positive for carbapenemase production but negative for metallo-beta-lactamase (MBL) production. Single patient isolates were tested by Vitek 2, Etest, agar dilution MICs, phenotypic assays and PFGE. Carbapenemase and other beta-lactamase genes were identified by PCR and sequencing. Patient records were retrospectively reviewed to access co-morbidities, antibiotic exposure prior to infection and outcome. RESULTS: The 47 K. pneumoniae isolates exhibited various susceptibilities to imipenem and meropenem; all were non-susceptible to ertapenem and several other antibiotics but most were susceptible to gentamicin, colistin and tigecycline. PFGE classified the isolates into two clonal types, with the predominant type, which was closely related to that of hyperepidemic strains from the USA and Israel, comprising three subtypes. All isolates carried the bla(KPC-2) gene; 45 also carried bla(SHV-12) and 29 bla(TEM-1). Patients were hospitalized in nine different units. The median length of hospital stay prior to KPC isolation was 21 days; 38 patients (80.9%) had evidence of clinical infection due to a KPC producer and 16 (34%) had bacteraemia. The crude mortality rate was 27.7%. A beta-lactam/beta-lactamase inhibitor combination was the most frequently administered antimicrobial prior to KPC isolation (20 patients; 42.5%), whereas only nine patients (19.1%) had prior carbapenem use. CONCLUSIONS: This study presents for the first time a wide intrahospital spread of KPC-producing K. pneumoniae clones in a European hospital. The KPC producers were rapidly disseminated in several units, indicating the difficulty in restraining such multidrug-resistant clones when they have been established in a hospital environment.

Cord blood survivin concentrations in human full-term normal and complicated pregnancies.

BACKGROUND: Survivin (a member of the inhibitors of apoptosis family) is important for fetal development, placental survival and differentiation. Intrauterine growth restriction (IUGR) and fetal macrosomia, due to maternal diabetes mellitus (DM) are associated with excessive and decreased feto-placental apoptosis, respectively. The aim was to study survivin concentrations in cord blood at term in IUGR, large-for-gestational-age (LGA, due to gestational DM) and appropriate-for-gestational-age (AGA) pregnancies. PATIENTS AND METHODS: Survivin concentrations were determined in 160 mixed arterio-venous cord blood samples from IUGR (n=48), LGA (n=11) and AGA (n=101) singleton full-term infants. RESULTS: No significant differences in survivin concentrations in cord blood were observed between groups. The effect of birthweight, customized centile, gestational age, gender, delivery mode and parity on survivin concentrations was not significant. CONCLUSION: Survivin concentrations in cord blood at term are independent of intrauterine growth, gender, parity and delivery mode. Thus, they probably do not reflect the disturbances of feto-placental apoptosis expected in IUGR and fetal macrosomia due to gestational DM.

Cytokine concentrations during the first days of life.

OBJECTIVE: To evaluate the cytokine concentration patterns during the first 5 days of life by measuring serum concentrations of type-1 cytokines, like interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) and type-2 cytokines, like IL-4, as well as the receptors of IL-2 (sIL-2R) and IL-4 (sIL-4R) during the early neonatal period. SUBJECTS AND METHODS: Forty-two healthy term neonates were included in the study. Cytokine concentrations were measured in umbilical cord, in the 1st and 5th day after birth and compared with those in serum of 30 healthy adults. RESULTS: IL-2 concentrations presented a decrease trend from umbilical cord to 5th day, while sIL-2R showed a significant elevation from umbilical cord to 5th day after birth. IL-4 concentrations did not differ significantly among umbilical cord, the 1st and the 5th day, while the sIL-4R showed the highest values in the 1st day after birth. Both IL-4 and sIL-4R concentrations in neonatal samples were elevated compared to adults. IFN-gamma concentrations increased significantly from umbilical cord to 5th day of life. CONCLUSION: Our findings indicate a dysregulation among IL-2, IL-4 and IFN-gamma concentrations during the 1st day after birth, favoring a more precocious expression of IL-2 and IL-4 against IFN-gamma that seems to be ameliorated in the end of the 1st week of life.

The influence of the mode of delivery on circulating cytokine concentrations in the perinatal period.

BACKGROUND: Cytokines play an important role during labor and full- or preterm delivery. They influence physical immunity of the fetus-neonate and express a leading role in the perinatal period, being present in maternal and fetal tissues. AIM: To investigate whether cytokine concentrations in the mother, fetus and neonate depend on the labor and the mode of the delivery. STUDY DESIGN: Prospective study. SUBJECTS: Seventy-eight healthy, non-smoking parturients (mean age 28+/-4, range 21-39 years, delivering vaginally: n=52 or by elective cesarean section: n=26) and their single, healthy, appropriate for gestational age, full-term neonates. OUTCOME MEASURES: We correlated determined circulating levels of IL-2, sIL-2R, IL-4, sIL-4R, IL-6, sIL-6R, IL-1beta, IL-8, IFN-gamma, TNF-alpha, sTNF RI, sTNF RII and RANTES in the mothers before delivery (MS), the fetuses (UC) and the neonates in days 1 (N1) and 4 (N4) of life, with the mode of delivery. RESULTS: sIL-2R in N1 and N4, sIL-4R in MS, IL-6 in MS and UC, IL-1beta in MS, UC and N1, IFN-gamma in MS and UC, TNF-alpha in UC, N1 and N4, sTNF RI in UC were significantly higher in cases of vaginal delivery than in cases of elective cesarean section (p ranging from 0.0005 to 0.05). CONCLUSIONS: Vaginal delivery promotes the production of various cytokines and their receptors, which are implicated in neonatal immunity.

Concentrations of soluble Fas in maternal serum and amniotic fluid during uncomplicated pregnancies.

OBJECTIVE: To determine, during normal pregnancy, maternal serum (MS) and amniotic fluid (AF) concentrations of soluble Fas (sFas), an apoptosis-suppressing molecule that might play a role in the apoptotic process. Soluble Fas levels might explain existing immunotolerance, fetal well being, and rupture of membranes at term. METHODS: Sixty-six healthy, nonsmoking, pregnant women (mean age 32.6 +/- 4.8 years) with uncomplicated singleton pregnancies (15 in the first trimester, 30 in the second trimester, and 21 at term vaginal delivery) and 20 healthy nonpregnant women (mean age 32.5 +/- 3.8 years) were included in the study. RESULTS: MS and AF sFas concentrations were measured by a sandwich enzyme-linked immunosorbent assay, and parametric tests were used in the statistical analysis.MS and AF sFas concentrations significantly depended on gestational age (P < .0008 and P < .0002, respectively). MS concentrations were significantly lower in the first trimester than those in the second trimester (P <.003), those at term (P < .03), and those in nonpregnant controls (P < .005). AF concentrations decreased significantly at term compared with those in the second trimester (P < .0003). AF sFas concentrations in the second trimester and at term were significantly lower than respective MS concentrations (P < .00001). CONCLUSION: MS sFas concentrations decreased significantly in the first trimester of pregnancy, possibly affecting semiallograft tolerance. In the second trimester, concentrations return to control levels and remain unchanged until delivery. AF sFas concentrations decrease at term compared with the second trimester, possibly indicating increased apoptosis in preparation for rupture of membranes.

Soluble Fas antigen and soluble Fas ligand in early neonatal life.

BACKGROUND: After birth, apoptosis rates might slow down, compared to those in utero. Thus, factors, attenuating the apoptotic process, like the soluble forms of Fas/FasL system, may increase. AIM-STUDY DESIGN: Soluble Fas (sFas) and soluble Fas ligand (sFasL) concentrations were measured in maternal serum (MS), umbilical cord (UC) and neonatal serum in the first (1N) and fifth (5N) days after birth in order to evaluate the alterations of these molecules during the early neonatal period. SUBJECTS AND METHODS: Soluble molecules were estimated in 35 healthy, appropriate for gestational age, full-term neonates, their mothers and in 25 healthy, nonpregnant women, age-matched to the mothers (controls), using enzyme immunoassays. RESULTS: sFas concentrations in MS (p < 0.01), UC (p < 0.0001), 1N (p < 0.0003) and 5N (p < 0.02) were lower than those in controls. Neonatal sFas concentrations showed a significant increase from UC to 5N (p < 0.001). In contrast, sFasL concentrations were significantly elevated in all neonatal samples (UC, 1N and 5N) compared to those in MS and controls (p < 0.0001), showing also a significant elevation from UC to 5N (p < 0.0001). CONCLUSION: Our results demonstrate increasing serum concentrations of the soluble molecules sFas and sFasL during the first days after birth, indicating possibly a gradual decrease of apoptosis in early neonatal life.

Mucin-like carcinoma-associated antigen in paired serum and breast milk samples of lactating mothers and sera of their neonates in the early postpartum period.

OBJECTIVE: To evaluate postpartum MCH changes in the early postpartum period, and to examine whether neonatal MCA is related to that in maternal serum (MS) or milk. STUDY DESIGN: MCA was measured by EIA on the second and fifth postpartum day in serum and BM from 30 lactating women and their single term neonates. Sera from 20 healthy women (controls), were also analyzed. RESULTS: All neonatal antigen concentrations were below the cut-off level for MCA (11 ng/ml). MS MCA was significantly increased compared with that in controls (P<0.00001), while antigen values in BM were highly elevated (P<0.00001), with a significant increase (P<0.0003) from the second to the fifth postpartum day. A strong correlation was found between the second and fifth day postpartum samples in MS, neonatal serum and BM MCA concentrations (r(s)=0.94, P<0.00001; r(s)=0.75, P<0.00001 and r(s)=0.69, P<0.0001, respectively). A significant correlation was also found in MCA values on the fifth postpartum day between neonatal serum and BM (r(s)=0.54, P<0.02). CONCLUSIONS: From these findings one may speculate on some ripening process in milk production and a possible transition of MCA from the neonatal gastro-intestinal tract into circulation.

Vancomycin-resistant Enterococcus outbreak in a neonatal intensive care unit: epidemiology, molecular analysis and risk factors.

BACKGROUND: Vancomycin-resistant Enterococcus faecium (VRE) may cause outbreaks in neonatal intensive care units (NICU). We describe a biphasic VRE outbreak and identify risk factors for VRE acquisition. METHODS: After the occurrence of 2 cases of VRE infections in a 44-bed NICU, a bundle of interventions was implemented that included active surveillance cultures for VRE, enhanced infection control measures, and audits on antimicrobial use, from June to December 2008. Analysis was performed using polymerase chain reaction and pulse-field gel electrophoresis techniques. A case-control study was conducted to identify risk factors. RESULTS: Among 253 neonates screened, 101 (39.9%) were found to be colonized with VRE. During the first 9 weeks of the study period, 59 new cases were detected. Molecular analysis showed 1 predominant clone. During weeks 10-12, no new cases of VRE colonization were detected; however, at week 13, just when the outbreak appeared to be over, a second wave occurred, with 42 new cases and multiple clones detected. Multivariate analysis identified administration of antimicrobial therapy for late-onset neonatal sepsis and hospitalization during the first month of this outbreak as significant risk factors for VRE colonization. CONCLUSION: Both a high prevalence of VRE colonization and antimicrobial use promoted the transmission of VRE during this biphasic outbreak. Adherence to infection control measures and antimicrobial stewardship policies are of utmost importance.

Post‒cataract surgery endophthalmitis outbreak caused by multidrug-resistant Pseudomonas aeruginosa.

In June 2010, a severe outbreak of 13 cases of post-cataract surgery endophthalmitis caused by multidrug-resistant Pseudomonas aeruginosa occurred. Pulse-field gel electrophoresis in eye isolates found 95% genetic similarity; however, extensive environmental and carriage investigation revealed no source of infection.

Tumor markers in biological fluids associated with pregnancy.

Proteins that are expressed by both malignant and healthy fetal tissues are recognized as oncofetal. These antigens are associated with cell proliferation and differentiation and are produced in high concentrations in pregnancy and malignancy. Their biological role in malignancy is the suppression of the host's immune system, while in pregnancy they affect the maternal immune response, generating maternal tolerance toward the embryo. This review describes the levels of alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), carcinoembryonic antigen (CEA), cancer antigen 125 (CA 125), squamous cell carcinoma antigen (SCC), cancer antigen 15-3 (CA 15-3), mucin-like carcinoma-associated antigen (MCA), tissue polypeptide-specific antigen (TPS), carbohydrate antigen 19-9 (CA 19-9), and prostate-specific antigen (PSA) in maternal serum (MS), umbilical cord serum (UC), and amniotic fluid (AF) and outlines their roles in the assessment of pregnancy and malignancy. All antigens studied, except CA 15-3, are oncofetal. The presence of considerable concentrations of AFP, hCG, CEA, CA125, SCC, MCA, TPS, CA 19-9, and PSA in AF during pregnancy may be attributed to their involvement in biological functions associated with fetal development, differentiation, and maturation. MS CEA, CA 15-3, and CA 19-9, in contrast to all the others, are not influenced significantly by pregnancy and thus remain reliable tumor markers in monitoring malignancy in pregnant patients.

Indicative markers of cell proliferation and apoptosis during the perinatal period.

Tissue polypeptide-specific antigen (TPS), an indicative marker of cell proliferation and soluble Fas (sFas), an antiapoptotic molecule were determined in neonatal serum (day 1-N1 and day 4-N4 of life), compared with maternal serum (MS) and umbilical cord serum (UC) to study changes of these markers during the perinatal period. Serum TPS and sFas concentrations were measured in 33 healthy, termed neonates, their mothers and 25 healthy nonpregnant controls (CS), age-matched to the mothers. TPS serum concentrations were significantly elevated in N1 and N4 as compared to CS (p < 0.0001 and p < 0.0003), increasing significantly from UC to N1 (p < 0.0001) and decreasing thereafter in N4 (p < 0.0002). MS serum concentrations, being significantly higher than CS (p < 0.0001), UC (p < 0.0001) and N4 (p < 0.003), but lower than N1 (p < 0.02) were strongly depended on the mode of delivery (p < 0.001). Serum concentrations of sFas, being lower in UC than in MS or CS (p < 0.0001), increased significantly in N4 samples (p < 0.01). A strong correlation was found between sFas serum concentrations in N1 and N4 (r = 0.65; p < 0.001). Our results demonstrate significant perinatal changes in TPS and sFas serum concentrations, possibly indicating gradual decrease of proliferation and apoptosis in early postnatal life.

Cytokine soluble receptors in perinatal and early neonatal life.

BACKGROUND: In contrast to cellular receptors, soluble receptors do not enhance the cellular activation because they do not have transmembranic and cytoplasmic parts, acting thereby as endogenous regulatory mechanisms against systemic functions of cytokines. AIM: To measure serum concentrations of the soluble interleukin-2 receptor (sIL2R), soluble interleukin-4 receptor (sIL4R), soluble interleukin-6 receptor (sIL6R), and soluble tumor necrosis factor-alpha receptor I and soluble tumor necrosis factor-alpha receptor II, during the perinatal and early neonatal period, in order to evaluate their role in activation of immune response in labor and the first days postpartum. METHODS: Soluble receptor serum concentrations were determined by enzyme-linked immunosorbent assay, in 45 healthy, full-termed neonates during the first and fifth days after birth, in 25 of their mothers (MS), in 25 samples of umbilical cords (UC) and in 25 healthy adult donors age-matched with the mothers (controls). RESULTS: Soluble receptor serum concentrations showed considerable changes during labor and early neonatal life, being significantly higher both in MS (except sIL6R) and in neonatal sample UC, first and fifth days after birth, compared with controls (p<0.0001). Neonatal serum sIL2R and sIL6R increased significantly from birth to the fifth day, while the remaining receptors showed a rapid increase in the first day (p<0.0001), declining significantly thereafter (p<0.0001). CONCLUSION: Our findings suggest that the elevated concentrations of all studied soluble cytokine receptors reflect the activation of immune response, and represent also regulatory protective mechanisms for mother and fetus-neonate against the systemic function of cytokines during labor and early neonatal life.