In vitro synergistic activity of erythromycin and nisin against clinical Group B Streptococcus isolates.

AIMS: This study investigated the potential synergy between erythromycin and nisin against clinical Group B Streptococcus (GBS) strains. METHODS AND RESULTS: The combination of erythromycin and nisin was examined for synergistic activity using checkerboard and time-kill assays against invasive and colonizing GBS strains. Additionally, the immunological effect of the antibiotic combination was investigated in vitro using human U937 cells and ELISA analysis. Checkerboard assays confirmed an additive effect when the antimicrobials were combined, while time-kill assays demonstrated a synergistic effect when antimicrobials were combined for invasive GBS isolates. Furthermore, a significantly lower TNF-alpha response (P < 0·05) was observed in U937 cells challenged with GBS when erythromycin and nisin were used in combination. CONCLUSIONS: The results suggest that erythromycin and nisin can act synergistically to inhibit the growth of GBS. SIGNIFICANCE AND IMPACT OF THE STUDY: Group B Streptococcus is the leading cause of invasive neonatal disease worldwide and is becoming increasingly more prevalent in adults. Resistance to some conventionally used antibiotics, such as erythromycin and clindamycin, continue to rise among GBS, indicating a need for alternative treatments. This study demonstrates the potential of an erythromycin-nisin combination for treatment of GBS infections and encourages further investigation of this treatment option.

A novel bioengineered derivative of nisin displays enhanced antimicrobial activity against clinical Streptococcus agalactiae isolates.

OBJECTIVES: Streptococcus agalactiae is the leading cause of neonatal disease worldwide, and infections caused by this opportunistic pathogen are becoming increasingly more prevalent in adults. With the global incidence of antimicrobial resistance continuing to rise, there is a recognised need for new therapeutic agents. Nisin is a potent antimicrobial peptide with demonstrated broad-spectrum activity against a range of clinically significant pathogens. This study aimed to examine the efficacy of nisin against a clinical population of S. agalactiae isolates and further to investigate the bioactivity of a novel bioengineered derivative of the peptide, designated nisin PV. METHODS: A deferred antagonism assay was used to assess the bioactivity of wild-type nisin and nisin PV against 122 S. agalactiae isolates. Minimum inhibitory concentrations (MICs) were evaluated to determine the specific activity of both peptides. The genetic basis of nisin resistance among the isolate collection was investigated by PCR detection of the nsr gene. RESULTS: In total, 91.0% (111/122) of the collection showed some level of susceptibility to nisin, whilst 9.0% (11/122) displayed complete resistance. Interestingly, the nisin derivative exhibited enhanced antimicrobial activity for 64.8% of the isolates. The frequency of the nsr gene conferring nisin resistance was 98.4% (120/122), suggesting that resistance may be linked to levels of expression of the protein or other regulatory elements. CONCLUSION: This study indicates that there is potential for the use of nisin and its derivatives as therapeutic agents against S. agalactiae infections.

Emergence of the L phenotype in Group B Streptococci in the South of Ireland.

Group B Streptococcal isolates (n = 235) from the South of Ireland were characterised by serotyping, antimicrobial susceptibility and determination of the phenotypic and genotypic mechanisms of resistance. Resistance to erythromycin and clindamycin was observed in 21·3% and 20·4% of the total population, respectively. The c-MLSB phenotype was the most common phenotype detected (62%), with ermB being the predominant genetic determinant, present in 84% of resistant isolates. The rare L phenotype was observed in 2·9% (n = 7) of isolates, four of which harboured the lsaC gene responsible for clindamycin resistance. Serotypes Ia, III and II were the most common amongst the entire study population (28·1%, 24·7% and 14%, respectively). Four of the seven L phenotype isolates were serotype III and two of these strains were confirmed as the hypervirulent clone, ST-17 and harboured the hvgA gene. This is the first documented case of the L phenotype in Ireland to date and the study findings emphasise the need for continued monitoring of antibiotic resistance and serotype distribution in GBS isolates from Ireland.

Bovine lactoferrin (LTF) gene promoter haplotypes have different basal transcriptional activities.

Genetic polymorphisms present in the bovine lactoferrin (LTF) gene promoter have the potential to affect milk lactoferrin concentrations. The objectives were: (1) to identify, in silico, SNPs in the promoter region of the LTF gene that could affect transcription factor binding activity, (2) to investigate the effects of these SNPs in vitro by measuring promoter transcriptional activities of different bovine LTF promoter haplotypes and (3) to investigate the genetic association between LTF promoter SNPs and milk lactoferrin concentration. Haplotypes were deduced from sequencing of the 2.2-kb bovine LTF promoter in 78 unrelated animals. In silico analysis of the 2.2-kb promoter revealed two major haplotypes (BtLTF_H1a and BtLTF_H2a) that differed at 10 SNP loci that affect transcription factors of both a constitutive (at -28, -1702) and an inducible (at -131, -270, -586, -2047, -2077, -2122, -2140 and -2151) nature. The basal promoter transcriptional activity of BtLTF_H1a was 1.44-fold higher than that of BtLTF_H2a in mammary epithelial cells. Cows with the BtLTF_H1a haplotype had increased lactoferrin protein concentration in milk at various time points over the lactation curves, compared to herdmates with the BtLTF_H2a haplotype. The SNPs c.-28A>C, c.-131T>C, c.-156A>G, c.-270T>C, c.-586C>T, c.-1702A>G, c.-1953G>A, c.-2047A>G, c.-2077A>G, c.-2122C>T, c.-2140A>G and c.-2151G>A were associated (P < 0.001) with milk lactoferrin content in 372 Holstein-Friesian cows. The identification of bovine LTF promoter haplotypes with different basal transcriptional activities in vitro that are associated with lactoferrin levels in milk in vivo may facilitate the identification of designer dairy herds for increased lactoferrin content in milk.

Polymorphisms in the bovine lactoferrin promoter are associated with reproductive performance and somatic cell count.

Lactoferrin plays an important role in the innate immune system, with well-characterized antibacterial, antiviral, and immune modulatory properties. The objective of this study was to determine the allele and haplotype frequency of polymorphisms at positions -586, -190, and -28 of the bovine lactoferrin promoter in Holstein-Friesians and to quantify their association with performance using phenotypic data on progeny from 848 sires. Associations between genotypes and performance were quantified using weighted mixed models with genotyped individuals included as a random effect, and average expected relationships among individuals accounted for through a numerator relationship matrix. The dependent variables were daughter yield deviation for production traits and deregressed predicted transmitting ability for calving interval and functional survival. The C to T polymorphism at -586, which distorts a putative activating protein 2 (AP-2) binding site, was associated with a shorter calving interval and higher somatic cell score. The G to A polymorphism at -190, located in a putative selective promoter factor 1 (SP-1) binding site, was associated with a longer calving interval and decreased functional survival. A third polymorphism (A to C) at position -28, found within the noncanonical TATA box, had a tendency to associate with functional survival. On the basis of the data we proposed a haplotype combination that was associated with improved reproductive performance in the Holstein-Friesian breed. We hypothesized that the observable phenotypic associations with lactoferrin promoter polymorphisms can potentially be explained by allele-specific differences in constitutive or inducible levels of gene expression. The lack of a pleiotropic effect of the single nucleotide polymorphisms studied on both fertility and milk production traits strengthens the importance of these polymorphisms, or at least the lactoferrin promoter, in selection for improved fertility.

Characterisation of single nucleotide polymorphisms identified in the bovine lactoferrin gene sequences across a range of dairy cow breeds.

The lactoferrin gene sequences of 70 unrelated dairy cows representing six different dairy breeds were investigated for single nucleotide polymorphisms to establish a baseline of polymorphisms that exist within the Irish bovine population. Twenty-nine polymorphisms were identified within a 2.2kb regulatory region. Nineteen novel polymorphisms were identified and some of these were found within transcription factor binding sites, including GATA-1 and SPI transcription factor sites. Forty-seven polymorphisms were identified within exon sequences with unique polymorphisms that were associated with amino acid substitutions. These included a T/A SNP, identified in a Holstein Friesian animal, which resulted in a valine to aspartic acid substitution (Val89Asp) in the mature lactoferrin protein. Other SNPs of interest were associated with amino acid substitutions in the lactoferricin B peptide sequence and an A/G SNP, identified in a Jersey animal, was associated with a tyrosine to cysteine change (Tyr181Cys). The polymorphisms identified in the promoter region may have implications relating to lactoferrin expression levels in cows and those identified in the coding sequence indicate the existence of protein variants in the Irish bovine population. The data presented in this study emphasises the potential for lactoferrin to serve as a candidate gene to select for mastitis resistance with the aim of improving animal health.

Phenotypic and genotypic anti-microbial resistance profiles of campylobacters from untreated feedlot cattle and their environment.

Anti-microbial resistance is an emerging public health issue. Farmed animals may act as reservoirs and potential sources of anti-microbial resistant Campylobacters. The aim of this study was to investigate the anti-microbial resistance profile of cattle and environmental Campylobacter isolates from normal untreated feedlot cattle, the role of the gyrA Thr-86-Ile mutation in ciprofloxacin-resistant Campylobacter jejuni isolates and the involvement of the tripartite CmeABC efflux system for multi-resistant C. jejuni isolates. The phenotypic anti-microbial resistance testing was carried out on 500 Campylobacter isolates (445 cattle isolates and 55 environmental isolates). In general, there was a higher level of anti-microbial resistance for the environmental isolates compared with the animal isolates, 45% of the animal isolates were resistant to one or more of the seven anti-microbials compared with 84% of the environmental isolates. The combined cattle and environmental Campylobacters had 34 (6.8%) isolates resistant to three or more of the seven anti-microbials tested on all isolates and 11 (2.2%) isolates were resistant to the seven anti-microbials. There was a substantial level of ciprofloxacin-resistant Campylobacters in both animal (8.5%) and environmental (21.8%) isolates. The gyrA Thr-86-Ile mutation was only present in five of 22 ciprofloxacin-resistant C. jejuni isolates investigated. No multi-drug-resistant associated mutation was detected in the CmeB or the CmeR regions investigated. In conclusion, our study observed a substantial level of Campylobacter anti-microbial resistance, highlighting the need for an active anti-microbial surveillance program for food animals in Ireland and the importance of the chosen sampling point can have on the findings of such a program.

Emergence of G3 and G9 rotavirus and increased incidence of mixed infections in the southern region of Ireland 2001-2004.

Two hundred and thirty fecal specimens were collected from children (up to 5 years of age) admitted with suspected rotaviral gastroenteritis at four Irish hospitals (Cork University Hospital, Mercy Hospital, Cork, Waterford Regional Hospital, and Kerry General Hospital) in the southern region of Ireland, between 2001 and 2004. Following laboratory confirmation of the aetiological agent, the rotavirus G-type was determined in all positive samples by reverse transcriptase-polymerase chain reaction (RT-PCR). The distribution of the G-types (n=230) over the 3 year period was G1 (31%), G9 (21.8%), G3 (8.7%), G4 (6.5%), and G2 (3.5%). There were many mixed infections which accounted for 28.5% of the collection. G9 emerged as the most prevalent G type (30.1%) in 2001-2002, whilst G3 first emerged in 2002-2003 and accounted for 15.8% of the collection. Notably, G2 strains were present at a very low frequency (3.5%) during 2001-2004, compared to an earlier study (1997-1999), where they accounted for 28.5% of the specimens. A smaller subset of the study collection was similarly P-typed (n=139). P[8]-type was identified as the most prevalent P-type, accounting for 97.4% (n=186), while P[4] accounted for just 2.6% (n=5) of the collection. The low frequency of P[4] coincided with the decrease in G2 strains in circulation. The key finding in this study was the emergence of G3- and G9-serotypes as epidemiologically important rotavirus strains since 1999, and the low prevalence of the previously common G2 strains in Ireland. The profile of rotavirus is changing continuously in Ireland and the implications for a successful vaccination program are discussed.

Trends in antimicrobial susceptibility among isolates of Campylobacter species in Ireland and the emergence of resistance to ciprofloxacin.

Measurements were made of the susceptibility to six commonly prescribed antibiotics, including erythromycin, tetracycline and ciprofloxacin, of 130 isolates of Campylobacterjejuni and 15 isolates of Campylobacter coli cultured from human and poultry sources during 2000. The results were compared with the results from a collection of strains isolated between 1996 and 1998. The levels of resistance to erythromycin remained low, 2 per cent and 4.4 per cent for the human and poultry isolates, respectively. Resistance to tetracycline had increased to 31 per cent and 24.4 per cent from 13.9 per cent and 18.8 per cent for the human and poultry isolates, respectively. However, the resistance to ciprofloxacin of the strains isolated during 2000 had increased to 30 per cent, whereas between 1996 and 1998 there had been no resistance to this agent among human isolates, and only 3.1 per cent resistance among poultry isolates. The molecular basis for this resistance has been shown to be the result of a single amino acid substitution, Thr-86-Ile, in the gyrA subunit of DNA gyrase in Cjejuni. A subset of 59 isolates was tested by molecular methods and all of the 25 phenotypically resistant isolates possessed this substitution. None of the human isolates had been treated with ciprofloxacin before their laboratory isolation.

Application of restriction fragment length polymorphism analysis of VP7-encoding genes: fine comparison of Irish and global rotavirus isolates.

A restriction fragment length polymorphism (RFLP) detection assay was developed to examine the genetic relationship(s) among VP7-encoding genes from 100 Irish rotavirus isolates and 30 randomly selected global rotavirus isolates (from the current databases). RFLP analysis of the VP7 gene segments was performed independently with three enzymes (RsaI, AluI, and EcoRV) in separate reactions by direct digestion of the DNA product amplified by reverse transcriptase (RT)-mediated PCR (RT-PCR) or by using computational methods. Thirty-six RFLP patterns were identified for all 130 strains, and of these, only nine patterns were associated with the Irish isolates. A correlation between the G type of the Irish isolates and certain single or combined enzyme profiles was apparent. These data suggested that the Irish wild-type rotavirus population was homogeneous and could be distinguished by RFLP analysis from global isolates of the same serotype(s). The deduced amino acid sequences of the VP7 RT-PCR products from six Irish isolates known to be of the G serotype revealed significant amino acid substitutions within major antigenic regions. In addition, these data identified the existence of at least two genetic lineages within serotype G1 strains which were distinguishable by RFLP analysis.

Rotavirus in Ireland: national estimates of disease burden, 1997 to 1998.

BACKGROUND: We estimated the disease burden caused by rotavirus hospitalizations in the Republic of Ireland by using national data on the number of hospitalizations for diarrhea in children and laboratory surveillance of confirmed rotavirus detections. METHODS: We examined trends in diarrheal hospitalizations among children <5 years old as coded by ICD-9-CM for the period January, 1997, to December, 1998. We collated data on laboratory-confirmed rotavirus detections nationally for the same period among children <2 years old. We calculated the overall contribution of rotavirus to laboratory-confirmed intestinal disease in children <5 years old from INFOSCAN, a disease bulletin for one-third of the population. We compared data from all sources and estimated the proportion of diarrheal hospitalizations that are likely the result of rotavirus in children <5 years old. RESULTS: In children <5 years old, 9% of all hospitalizations are for diarrheal illness. In this age group 1 in 8 are hospitalized for a diarrheal illness, and 1 in 17 are hospitalized for rotavirus by 5 years of age. In hospitalized children <2 years old, 1 in 38 have a laboratory confirmed rotavirus infection. CONCLUSIONS: The disease burden of rotavirus hospitalizations is higher than in other industrialized countries. Access to comprehensive national databases may have contributed to the high hospitalization rates, as well as a greater tendency to hospitalize children with diarrhea in Ireland.

Molecular characterization of rotavirus in Ireland: detection of novel strains circulating in the population.

A collection of three hundred thirty rotavirus-positive stool samples from children with diarrhea in the southern and eastern regions of Ireland between 1997 and 1999 were submitted to the Molecular Diagnostics Unit of the Cork Institute of Technology, Cork, Ireland, for investigation. These strains were characterized by several methods, including polyacrylamide gel electropherotyping and G and P genotyping. A subset of the G types was confirmed by nucleic acid sequencing. The most prevalent types found in this collection included G1P[8] (n = 106; 32.1%), G2P[4] (n = 94; 28.5%), and G4P[8] (n = 37; 11.2%). Novel strains were also detected, including G1P[4] (n = 19; 5.8%), and G4P[4] (n = 2; 0.6%). Interestingly, mixed infections accounted for 18.8% (n = 62) of the total collection, with only 3% (n = 10) which were not G and/or P typeable. Significantly, six G8 and five G9 strains were identified as part of mixed infections. These strains have not previously been identified in Irish children, suggesting a greater diversity in rotavirus strains currently circulating in Ireland.

Integronlike structures in Campylobacter spp. of human and animal origin.

Resistance to antimicrobial agents used to treat severe Campylobacter spp. gastroenteritis is increasing worldwide. We assessed the antimicrobial resistance patterns of Campylobacter spp. isolates of human and animal origin. More than half (n = 32) were resistant to sulphonamide, a feature known to be associated with the presence of integrons. Analysis of these integrons will further our understanding of Campylobacter spp. epidemiology.

Use of immunobeads to detect human antispermatozoal antibodies.

Immunobeads for IgA, IgG and IgM were used in an indirect test (immunobead test: IBT) to detect human antispermatozoal antibodies, with positive results for at least one class of antibody being found in the serum of 13/169 (7.7%) men tested and 12/172 (6.9%) women. Of those men with antibodies present in serum, 100% had IgG, 62% had IgA and none had IgM, whilst the proportion for women was 75%, 100% and 33% respectively for each class of antibody. Antispermatozoal antibodies in men do not always appear both in semen and serum, but may be present in only one of the fluids tested for IgA (7/13 men; 53%) and IgG (6/14 men; 42.9%). The incidence of antibodies in the serum of oligospermic men was not significantly different from that of normospermic men (chi 2 = 0.06). A total of 481 serum and semen specimens were assayed by both the IBT and tray agglutination tests, and agreement between the two assays occurred in 97.3% (468/481) samples (P less than 0.001).

The value of the postcoital test in predicting the fertilization of human oocytes.

The relevance of the postcoital test (PCT) in relation to the fertilization rate of oocytes was determined in an analysis of 66 couples in an in vitro fertilization (IVF) program. The test is routinely performed in the workup of all IVF patients and is accurately timed to the immediate preovulatory period by daily hormonal, cervical mucus, and ultrasound monitoring. Cases demonstrating antispermatozoal antibodies in the serum of either partner, in cervical mucus, or in semen were excluded. There was no significant difference in the fertilization rate of oocytes whether the PCT result was negative, equivocal, or positive, and the finding was the same in both normospermic and oligospermic groups. It is concluded that the PCT is of no value in predicting the outcome of IVF and, conversely, IVF should be considered as a therapeutic option for couples with a negative PCT.