Enhanced antibiotic multi-resistance in nasal and faecal bacteria after agricultural use of streptomycin

Streptomycin is used in arboriculture to control fire blight. Using sheep as a model, multidrug-resistant bacteria in mammals were found to be selected after the intentional release of streptomycin into the environment. Escherichia coli and Staphylococcus spp. were isolated from the faeces and nasal cavities, respectively, of sheep grazing on a field sprayed with streptomycin at concentrations used in orchards (test group) and on a field without streptomycin (control group). Before the application of streptomycin, the percentage of streptomycin-resistant E. coli isolates in faeces was 15.8% in the control group and 14.7% in the test group. After the application of streptomycin, the overall number of streptomycin-resistant E. coli isolates was significantly higher in the test group (39.9%) than in the control group (22.3%). Streptomycin-resistant Staphylococcus isolates were only detected after the application of streptomycin. Streptomycin resistance was frequently associated with resistance to sulfamethoxazole, ampicillin, tetracycline and chloramphenicol and less frequently to cefotaxime in E. coli, and to tetracycline, fusidic acid and tiamulin in Staphylococcus spp. This study shows that the application of low concentrations of streptomycin on grass, as occurs during the spraying of orchards, selects for multidrug-resistant nasal and enteric bacterial flora, including extended-spectrum beta-lactamase-producing E. coli.

Non-phenotypic tests to detect and characterize antibiotic resistance mechanisms in Enterobacteriaceae

In the past 2 decades, we have observed a rapid increase of infections due to multidrug-resistant Enterobacteriaceae. Regrettably, these isolates possess genes encoding for extended-spectrum β-lactamases (e.g., blaCTX-M, blaTEM, blaSHV) or plasmid-mediated AmpCs (e.g., blaCMY) that confer resistance to last-generation cephalosporins. Furthermore, other resistance traits against quinolones (e.g., mutations in gyrA and parC, qnr elements) and aminoglycosides (e.g., aminoglycosides modifying enzymes and 16S rRNA methylases) are also frequently co-associated. Even more concerning is the rapid increase of Enterobacteriaceae carrying genes conferring resistance to carbapenems (e.g., blaKPC, blaNDM). Therefore, the spread of these pathogens puts in peril our antibiotic options. Unfortunately, standard microbiological procedures require several days to isolate the responsible pathogen and to provide correct antimicrobial susceptibility test results. This delay impacts the rapid implementation of adequate antimicrobial treatment and infection control countermeasures. Thus, there is emerging interest in the early and more sensitive detection of resistance mechanisms. Modern non-phenotypic tests are promising in this respect, and hence, can influence both clinical outcome and healthcare costs. In this review, we present a summary of the most advanced methods (e.g., next-generation DNA sequencing, multiplex PCRs, real-time PCRs, microarrays, MALDI-TOF MS, and PCR/ESI MS) presently available for the rapid detection of antibiotic resistance genes in Enterobacteriaceae. Taking into account speed, manageability, accuracy, versatility, and costs, the possible settings of application (research, clinic, and epidemiology) of these methods and their superiority against standard phenotypic methods are discussed.

Antibiotic prophylaxis for urinary tract infections after removal of urinary catheter: meta-analysis

Abstract Objective To determine whether antibiotic prophylaxis at the time of removal of a urinary catheter reduces the risk of subsequent symptomatic urinary tract infection. Design Systematic review and meta-analysis of studies published before November 2012 identified through PubMed, Embase, Scopus, and the Cochrane Library; conference abstracts for 2006-12 were also reviewed. Inclusion criteria Studies were included if they examined antibiotic prophylaxis administered to prevent symptomatic urinary tract infection after removal of a short term (≤14 days) urinary catheter. Results Seven controlled studies had symptomatic urinary tract infection after catheter removal as an endpoint; six were randomized controlled trials (five published; one in abstract form) and one was a non-randomized controlled intervention study. Five of these seven studies were in surgical patients. Studies were heterogeneous in the type and duration of antimicrobial prophylaxis and the period of observation. Overall, antibiotic prophylaxis was associated with benefit to the patient, with an absolute reduction in risk of urinary tract infection of 5.8% between intervention and control groups. The risk ratio was 0.45 (95% confidence interval 0.28 to 0.72). The number needed to treat to prevent one urinary tract infection was 17 (12 to 30). Conclusions Patients admitted to hospital who undergo short term urinary catheterization might benefit from antimicrobial prophylaxis when the catheter is removed as they experience fewer subsequent urinary tract infections. Potential disadvantages of more widespread antimicrobial prophylaxis (side effects and cost of antibiotics, development of antimicrobial resistance) might be mitigated by the identification of which patients are most likely to benefit from this approach.

From diarrhea to obesity in prohormone convertase 1/3 deficiency: age-dependent clinical, pathologic, and enteroendocrine characteristics

GOALS The aim of this report is to delineate the clinical, pathologic, and enteroendocrine (EE) features of prohormone convertase 1/3 (PC1/3) deficiency in children. BACKGROUND Prohormone convertases play a pivotal role in the activation of biologically inactive hormones. Congenital defects in the EE axis, such as PC1/3 deficiency, have been rarely reported and their pathophysiological mechanisms are largely unknown. STUDY EE function and pathology was evaluated in 4 males (1, 2, 7, and 10 y old) from 2 families with PC1/3 deficiency at a university children's hospital. Clinical course, pathology analysis including immunohistochemistry for PC1/3, PC2, and glucagon-like peptide 1 (GLP-1) and electron microscopy, as well as EE function tests (GLP-1, GLP-2, oral glucose tolerance test) were performed. RESULTS All (n=4) suffered from congenital severe diarrhea associated with malabsorption. The diarrhea improved during the first year of life and hyperphagia with excessive weight gain (BMI>97th percentile) became the predominant phenotype at an older age. Analysis of the enteroendocrine axis revealed high proinsulin levels (57 to 1116 pmol/L) in all patients, low serum GLP-2 levels, and impaired insulin and GLP-1 secretion after an oral glucose tolerance test at a young age, with improvement in 1 older child tested. Electron microscopy showed normal ultrastructure of enterocytes and EE cells. Immunohistochemistry revealed normal expression of chromogranin A, a marker of EE cells but markedly reduced immunostaining for PC1/3 and PC2 in all patients. CONCLUSIONS PC1/3 deficiency is associated with an age dependent, variable clinical phenotype caused by severe abnormalities in intestinal and EE functions. Serum level of proinsulin can be used as an effective screening tool.

Congenital proprotein convertase 1/3 deficiency causes malabsorptive diarrhea and other endocrinopathies in a pediatric cohort

BACKGROUND & AIMS Proprotein convertase 1/3 (PC1/3) deficiency, an autosomal-recessive disorder caused by rare mutations in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene, has been associated with obesity, severe malabsorptive diarrhea, and certain endocrine abnormalities. Common variants in PCSK1 also have been associated with obesity in heterozygotes in several population-based studies. PC1/3 is an endoprotease that processes many prohormones expressed in endocrine and neuronal cells. We investigated clinical and molecular features of PC1/3 deficiency. METHODS We studied the clinical features of 13 children with PC1/3 deficiency and performed sequence analysis of PCSK1. We measured enzymatic activity of recombinant PC1/3 proteins. RESULTS We identified a pattern of endocrinopathies that develop in an age-dependent manner. Eight of the mutations had severe biochemical consequences in vitro. Neonates had severe malabsorptive diarrhea and failure to thrive, required prolonged parenteral nutrition support, and had high mortality. Additional endocrine abnormalities developed as the disease progressed, including diabetes insipidus, growth hormone deficiency, primary hypogonadism, adrenal insufficiency, and hypothyroidism. We identified growth hormone deficiency, central diabetes insipidus, and male hypogonadism as new features of PCSK1 insufficiency. Interestingly, despite early growth abnormalities, moderate obesity, associated with severe polyphagia, generally appears. CONCLUSIONS In a study of 13 children with PC1/3 deficiency caused by disruption of PCSK1, failure of enteroendocrine cells to produce functional hormones resulted in generalized malabsorption. These findings indicate that PC1/3 is involved in the processing of one or more enteric hormones that are required for nutrient absorption.

Apoptosis in Bovine viral diarrhea virus (BVDV)-induced mucosal disease lesions: a histological, immunohistological, and virological investigation

Cattle persistently infected with a noncytopathic Bovine viral diarrhea virus (BVDV) are at risk of developing fatal "mucosal disease" (MD). The authors investigated the role of various apoptosis pathways in the pathogenesis of lesions in animals suffering from MD. Therefore, they compared the expression of caspase-3, caspase-8, caspase-9, and Bcl-2L1 (Bcl-x) in tissues of 6 BVDV-free control animals, 7 persistently infected (PI) animals that showed no signs of MD (non-MD PI animals), and 11 animals with MD and correlated the staining with the localization of mucosal lesions. Caspase-3 and -9 staining were markedly stronger in MD cases and were associated with mucosal lesions, even though non-MD PI animals and negative controls also expressed caspase-9. Conversely, caspase-8 was not elevated in any of the animals analyzed. Interestingly, Bcl-x also colocalized with mucosal lesions in the MD cases. However, Bcl-x was similarly expressed in tissues from all 3 groups, and thus, its role in apoptosis needs to be clarified. This study clearly illustrates ex vivo that the activation of the intrinsic, but not the extrinsic, apoptosis pathway is a key element in the pathogenesis of MD lesions observed in cattle persistently infected with BVDV. However, whether direct induction of apoptosis in infected cells or indirect effects induced by the virus are responsible for the lesions observed remains to be established.

Periodontal pathogens and associated factors in aggressive periodontitis: results 5-17 years after active periodontal therapy

OBJECTIVES To assess the association between presence of periodontal pathogens and recurrence of disease in patients with aggressive periodontitis (AgP) after active periodontal therapy (APT) and further influencing factors. MATERIAL & METHODS Microbiological samples were taken from 73 patients with AgP 5-17 years after APT at 292 sites (deepest site per quadrant). Real-time polymerase chain reactions were used to detect the periodontal pathogens Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola. Uni- and multivariate analyses evaluated the associations between pathogens and recurrence of disease, smoking and adjunctive antibiotic therapy. RESULTS At re-examination A. actinomycetemcomitans could be detected in six patients (8.2%), P. gingivalis in 24 (32.9%), T. forsythia in 31 (42.5%) and T. denticola in 35 (48.0%). Increased levels of T. forsythia and T. denticola at re-examination were significantly associated with recurrence of disease in multivariate analyses (OR: 12.72, p < 0.001; OR 5.55, p = 0.002 respectively). Furthermore, high counts of T. denticola were found in patients with increased percentage of sites with clinical attachment levels (CAL) ≥ 6 mm compared to those with low counts (13.8% versus 3.2%, p = 0.005). CONCLUSION In patients with recurrence of disease T. forsythia and T. denticola were detected more frequently and in higher counts. Furthermore, T. denticola was found more frequently in patients with increased CAL.

Nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) among Swiss veterinary health care providers: detection of livestock- and healthcare-associated clones.

We screened a total of 340 veterinarians (including general practitioners, small animal practitioners, large animal practitioners, veterinarians working in different veterinary services or industry), and 29 veterinary assistants for nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus pseudintermedius (MRSP) at the 2012 Swiss veterinary annual meeting. MRSA isolates (n = 14) were detected in 3.8 % (95 % CI 2.1 - 6.3 %) of the participants whereas MRSP was not detected. Large animal practitioners were carriers of livestock-associated MRSA (LA-MRSA) ST398-t011-V (n = 2), ST398-t011-IV (n = 4), and ST398-t034-V (n = 1). On the other hand, participants working with small animals harbored human healthcare-associated MRSA (HCA-MRSA) which belonged to epidemic lineages ST225-t003-II (n = 2), ST225-t014-II (n = 1), ST5-t002-II (n = 2), ST5-t283-IV (n = 1), and ST88-t186-IV (n = 1). HCA-MRSA harbored virulence factors such as enterotoxins, β-hemolysin converting phage and leukocidins. None of the MRSA isolates carried Panton-Valentine leukocidin (PVL). In addition to the methicillin resistance gene mecA, LA-MRSA ST398 isolates generally contained additional antibiotic resistance genes conferring resistance to tetracycline [tet(M) and tet(K)], trimethoprim [dfrK, dfrG], and the aminoglycosides gentamicin and kanamycin [aac(6')-Ie - aph(2')-Ia]. On the other hand, HCA-MRSA ST5 and ST225 mainly contained genes conferring resistance to the macrolide, lincosamide and streptogramin B antibiotics [erm(A)], to spectinomycin [ant(9)-Ia], amikacin and tobramycin [ant(4')-Ia], and to fluoroquinolones [amino acid substitutions in GrlA (S84L) and GyrA (S80F and S81P)]. MRSA carriage may represent an occupational risk and veterinarians should be aware of possible MRSA colonization and potential for developing infection or for transmitting these strains. Professional exposure to animals should be reported upon hospitalization and before medical intervention to allow for preventive measures. Infection prevention measures are also indicated in veterinary medicine to avoid MRSA transmission between humans and animals, and to limit the spread of MRSA both in the community, and to animal and human hospitals.

Genetic polymorphisms associated with the inflammatory response in bacterial meningitis.

BACKGROUND Bacterial meningitis (BM) is an infectious disease that results in high mortality and morbidity. Despite efficacious antibiotic therapy, neurological sequelae are often observed in patients after disease. Currently, the main challenge in BM treatment is to develop adjuvant therapies that reduce the occurrence of sequelae. In recent papers published by our group, we described the associations between the single nucleotide polymorphisms (SNPs) AADAT +401C > T, APEX1 Asn148Glu, OGG1 Ser326Cys and PARP1 Val762Ala and BM. In this study, we analyzed the associations between the SNPs TNF -308G > A, TNF -857C > T, IL-8 -251A > T and BM and investigated gene-gene interactions, including the SNPs that we published previously. METHODS The study was conducted with 54 BM patients and 110 healthy volunteers (as the control group). The genotypes were investigated via primer-introduced restriction analysis-polymerase chain reaction (PIRA-PCR) or polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) analysis. Allelic and genotypic frequencies were also associated with cytokine and chemokine levels, as measured with the x-MAP method, and cell counts. We analyzed gene-gene interactions among SNPs using the generalized multifactor dimensionality reduction (GMDR) method. RESULTS We did not find significant association between the SNPs TNF -857C > T and IL-8 -251A > T and the disease. However, a higher frequency of the variant allele TNF -308A was observed in the control group, associated with changes in cytokine levels compared to individuals with wild type genotypes, suggesting a possible protective role. In addition, combined inter-gene interaction analysis indicated a significant association between certain genotypes and BM, mainly involving the alleles APEX1 148Glu, IL8 -251 T and AADAT +401 T. These genotypic combinations were shown to affect cyto/chemokine levels and cell counts in CSF samples from BM patients. CONCLUSIONS In conclusion, this study revealed a significant association between genetic variability and altered inflammatory responses, involving important pathways that are activated during BM. This knowledge may be useful for a better understanding of BM pathogenesis and the development of new therapeutic approaches.

Virulence-associated gene pattern of porcine and human Yersinia enterocolitica biotype 4 isolates.

Yersinia enterocolitica 4/O:3 is the most important human pathogenic bioserotype in Europe and the predominant pathogenic bioserotype in slaughter pigs. Although many studies on the virulence of Y. enterocolitica strains have showed a broad spectrum of detectable factors in pigs and humans, an analysis based on a strict comparative approach and serving to verify the virulence capability of porcine Y. enterocolitica as a source for human yersiniosis is lacking. Therefore, in the present study, strains of biotype (BT) 4 isolated from Swiss slaughter pig tonsils and feces and isolates from human clinical cases were compared in terms of their spectrum of virulence-associated genes (yadA, virF, ail, inv, rovA, ymoA, ystA, ystB and myfA). An analysis of the associated antimicrobial susceptibility pattern completed the characterization. All analyzed BT 4 strains showed a nearly similar pattern, comprising the known fundamental virulence-associated genes yadA, virF, ail, inv, rovA, ymoA, ystA and myfA. Only ystB was not detectable among all analyzed isolates. Importantly, neither the source of the isolates (porcine tonsils and feces, humans) nor the serotype (ST) had any influence on the gene pattern. From these findings, it can be concluded that the presence of the full complement of virulence genes necessary for human infection is common among porcine BT 4 strains. Swiss porcine BT 4 strains not only showed antimicrobial susceptibility to chloramphenicol, cefotaxime, ceftazidime, ciprofloxacin, colistin, florfenicol, gentamicin, kanamycin, nalidixic acid, sulfamethoxazole, streptomycin, tetracycline and trimethoprim but also showed 100% antibiotic resistance to ampicillin. The human BT 4 strains revealed comparable results. However, in addition to 100% antibiotic resistance to ampicillin, 2 strains were resistant to chloramphenicol and nalidixic acid. Additionally, 1 of these strains was resistant to sulfamethoxazole. The results demonstrated that Y. enterocolitica BT 4 isolates from porcine tonsils, as well as from feces, show the same virulence-associated gene pattern and antibiotic resistance properties as human isolates from clinical cases, consistent with the etiological role of porcine BT 4 in human yersiniosis. Thus, cross-contamination of carcasses and organs at slaughter with porcine Y. enterocolitica BT 4 strains, either from tonsils or feces, must be prevented to reduce human yersiniosis.

Interferon-gamma and interleukin-4 mRNA expression by peripheral blood mononuclear cells from pregnant and non-pregnant cattle seropositive for bovine viral diarrhea virus

The acceptance of the fetal allograft by pregnant women and mice seems to be associated with a shift from a Th 1 dominated to a Th 2 dominated immune response to certain infectious agents. The goal of this study was to examine cytokine expression in peripheral blood mononuclear cells (PBMCs) from cattle immune to bovine viral diarrhea virus (BVDV) to determine whether pregnancy also has an influence on the type of immune response in this species. Forty-six heifers and cows between 14 months and 13 years of age were included in this study. Twenty-four were seropositive and 22 seronegative for BVDV. Eleven of the seropositive animals and 11 of the seronegative animals were in the eighth month of gestation, the remaining animals were virgin heifers. PBMC from these animals were analyzed for Interferon (IFN)-gamma and Interleukin (IL)-4 mRNA expression by real-time RT-PCR after stimulation with a non-cytopathic strain of BVDV. Additionally, an ELISA was performed to measure IFN-gamma in the supernatants of stimulated cell cultures. In BVDV seropositive animals, IFN-gamma mRNA levels were significantly higher than in BVDV seronegative animals and there was a significant positive correlation between the changes in IFN-gamma and IL-4 mRNA expression. There was, however, no significant difference in IFN-gamma and IL-4 mRNA levels between pregnant and non-pregnant animals. These results are inconsistent with BVDV inducing a Th1 or Th2 biased immune response. Furthermore, a shift in the cytokine pattern during bovine pregnancy was not evident.

Rapid Communication: Cholesterol deficiency-associated APOB mutation impacts lipid metabolism in Holstein calves and breeding bulls.

During the last months, the number of reports on Holstein calves suffering from incurable idiopathic diarrhea dramatically increased. Affected calves showed severe hypocholesterolemia and mostly died within days up to a few months after birth. This new autosomal monogenic recessive inherited fat metabolism disorder, termed cholesterol deficiency (CD), is caused by a loss of function mutation of the bovine gene. The objective of the present study was to investigate specific components of lipid metabolism in 6 homozygous for the mutation (CDS) and 6 normal Holstein calves with different genotypes. Independent of sex, CDS had significantly lower plasma concentrations of total cholesterol (TC), free cholesterol (FC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), very-low-density lipoprotein cholesterol (VLDL-C), triacylglycerides (TAG), and phospholipids (PL) compared with homozygous wild-type calves ( < 0.05). Furthermore, we studied the effect of the genotype on cholesterol metabolism in adult Holstein breeding bulls of Swissgenetics. Among a total of 254 adult males, the homozygous mutant genotype was absent, 36 bulls were heterozygous carriers (CDC), and 218 bulls were homozygous wild-type (CDF). In CDC bulls, plasma concentrations of TC, FC, HDL-C, LDL-C, VLDL-C, TAG, and PL were lower compared with CDF bulls ( < 0.05). The ratios of FC:cholesteryl esters (CE) and FC:TC were higher in CDC bulls compared with CDF bulls, whereas the ratio of CE:TC was lower in CDC bulls compared with CDF bulls ( < 0.01). In conclusion, the CD-associated mutation was shown to affect lipid metabolism in affected Holstein calves and adult breeding bulls. Besides cholesterol, the concentrations of PL, TAG, and lipoproteins also were distinctly reduced in homozygous and heterozygous carriers of the mutation. Beyond malabsorption of dietary lipids, deleterious effects of apolipoprotein B deficiency on hepatic lipid metabolism, steroid biosynthesis, and cell membrane function can be expected, which may result in unspecific symptoms of reduced fertility, growth, and health.