Comparison of antimicrobial resistant Enterobacterales isolates from the dairy production environment in low and high zinc containing regions.

Heavy metals occur naturally in the environment, and their concentration varies in soil across different regions. However, the presence of heavy metals may influence the antimicrobial resistance (AMR) in bacterial populations. Therefore, the objective of this study was to investigate and characterise the antimicrobial resistance profiles of Enterobacterales in soil and bovine milk filters from high and low zinc-containing regions in Ireland. In total, 50 soil samples and 29 milk filters were collected from two geographic locations with varying soil zinc concentrations. Samples were cultured for the enumeration and detection of Enterobacterales. Specifically, extended-spectrum beta-lactamase-producing Enterobacterales, carbapenem-resistant Enterobacterales and ciprofloxacin-resistant Enterobacterales were isolated using selective media. Species identification was performed using MALDI-TOF. The phenotypic resistance profiles of selected Enterobacterales were determined by disk diffusion testing, following EUCAST and CLSI criteria; while, the genotypic resistance profiles of the same isolates were determined by whole genome sequencing (WGS). Heavy metal concentrations were also measured for all soil samples. A total of 40 antimicrobial resistant Enterobacterales were identified in soil (n = 31) and milk filters (n = 9). The predominant species detected in the high zinc-containing region was Escherichia coli in both sample types (soil n = 10, milk filters n = 2), while in the low zinc-containing region Serratia fonticola was predominant in soil samples (n = 8) and E. coli in milk filters (n = 4). Ten E. coli isolates identified from soil samples in the high zinc-containing region were multidrug resistant, showing resistance to all the antimicrobials tested, except for carbapenems. The WGS findings confirmed the phenotypic resistance results. Moreover, zinc resistance-associated genes and genes encoding for efflux pumps were identified. The current study revealed distinct phenotypic resistance profiles of Enterobacterales in low and high zinc-containing regions, and highlighted the benefit of utilising milk filters for AMR surveillance in dairy production.

Characterization of antimicrobial resistant Enterobacterales isolated from spinach and soil following zinc amendment.

Antimicrobial resistant bacteria can occur in the primary food production environment. The emergence and dissemination of antimicrobial resistance (AMR) in the environment can be influenced by several factors, including the presence of heavy metals. The aim of this study was to examine the presence and characteristics of antimicrobial resistant Enterobacterales in soils and spinach grown in soils with and without zinc amendment. A total of 160 samples (92 soil and 68 spinach) were collected from two locations, in which some plots had been amended with zinc. Samples were cultured on selective agars for detection of extended-spectrum beta-lactamase-producing Enterobacterales (ESBL), carbapenem-resistant Enterobacterales and ciprofloxacin-resistant Enterobacterales. Samples were also cultured for enumeration of total Enterobacterales. Isolates were identified by MALDI-TOF. Antimicrobial susceptibility testing was carried out in accordance with EUCAST and CLSI criteria. The whole genome sequence (WGS) of selected isolates was determined. Inductively coupled plasma atomic emission spectrometry was also performed on soil samples in order to measure the concentration of zinc. In total 20 antimicrobial resistant Enterobacterales were isolated from the soil (n = 8) and spinach samples (n = 12). In both sample types, Serratia fonticola (n = 16) was the dominant species, followed by Escherichia coli (n = 1), Citrobacter freundii (n = 1) and Morganella morganii (n = 1) detected in spinach samples, and Enterobacter cloacae (n = 1) detected in a soil sample. The WGS identified genes conferring resistance to different antimicrobials in agreement with the phenotypic results; 14 S. fonticola isolates were confirmed as ESBL producers and harboured the blaFONA gene. Genes that encoded for zinc resistance and multidrug efflux pumps, transporters that can target both antimicrobials and heavy metals, were also identified. Overall, the findings of this study suggest the presence of zinc did not influence the AMR Enterobacterales in soil or spinach samples.

Examining the efficacy of mushroom industry biocides on Listeria monocytogenes biofilm.

AIMS: The aim of this study was to test the efficacy of new and currently used biocides in the mushroom industry for inactivating Listeria monocytogenes biofilm. METHODS AND RESULTS: A laboratory-scale study was initially carried out to test the efficacy of eleven biocidal products against a cocktail of five L. monocytogenes strains that were grown to 3-day biofilms on stainless steel coupons. Biocidal efficacy was then tested under clean and dirty conditions based on the EN 13697:2015 method. The results for the biocides tested ranged between 1·7-log and 6-log reduction of biofilm, with only the efficacy of the sodium hypochlorite-based biocide being significantly reduced in dirty conditions. A pilot-scale trial was then carried out on a subset of biocides against L. monocytogenes on concrete floors in a mushroom growing room and it was found that biocide efficacy in laboratory-scale did not translate well in pilot-scale. CONCLUSIONS: Biocides that are used in the mushroom industry and potential alternative biocides were determined to be effective against L. monocytogenes biofilm in both laboratory-scale and pilot-scale experiments. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has direct impact for the industry as it provides information on the efficacy of currently used biocides and other biocidal products against L. monocytogenes, an added benefit to their primary use.

Inactivation efficacy and mechanisms of plasma activated water on bacteria in planktonic state.

AIMS: The study aimed to investigate the inactivation efficacy and mechanisms of plasma activated water (PAW) on selected bacteria in planktonic state. METHODS AND RESULTS: Plasma activated water was generated using an atmospheric cold plasma jet at 15, 22 and 30 kV for 5 min. Escherichia coli, Listeria innocua, Staphylococcus aureus, Aeromonas hydrophila, Pseudomonas fluorescens and Shewanella putrefaciens were selected as the representative bacterial species. Each bacterial suspension was inoculated into PAW immediately after generation, and the viable counts at different exposure times of 0·5, 1, 3, 5 and 24 h during 4°C storage were measured to determine the inactivation efficacy. Scanning electron microscopy images of the bacteria were conducted to examine the structural changes. Physicochemical properties of PAW, including pH, conductivity, oxidation reduction potential (ORP), and reactive species of H2 O2 , NO2 - and NO3 - were measured. The results demonstrated that inactivation efficacy was in positive correlation with voltage and exposure time. Gram-negative bacteria were more susceptible to PAW than Gram-positive bacteria. Morphology damage was observed for all the bacterial species. PAW was significantly acidified, conductivity and ORP were significantly increased, and reactive species were detectable after 48 h. CONCLUSIONS: This study offered a better understanding of the inactivation mechanisms of PAW, and the inactivation efficacy can be affected by voltage, exposure time and bacterial species. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the potential usage of PAW as an alternative disinfectant.

Epithelial surface changes and spermatozoa storage in the reproductive tract of the bitch.

Spermatozoa are known to bind to the epithelial cells lining the uterine tube in various species, but information in canids is conflicting and sparse. The first aim of this study was to measure the epithelial surface outline (ESO) of different regions of the canine uterine tube in the four stages of the oestrous cycle as an indicator of a changing potential reservoir for spermatozoa. The second aim was to identify the site of sperm storage in the bitch after natural mating. Reproductive tracts were collected from bitches undergoing routine ovariohysterectomy. Histological analysis showed that, when corrected for uterine tube size, the ESO of pro-oestrus (P<0.005) and oestrus (P<0.05) tubes were larger than anoestrus, but not metoestrus, tubes. The second study examined reproductive tracts from 12 Beagle bitches at 6, 12, 24 and 48h after mating. Light and electron microscopy revealed large numbers of spermatozoa in the proximal regions of the uterus and particularly the distal utero-tubal junction (UTJ), with few present in the proximal UTJ or uterine tubes. Spermatozoa were bound by their heads to microvilli on the epithelial surface of the uterine lumen and to ciliated cells in the distal UTJ. This is the first report to measure and document differences in potential epithelial attachment sites of the uterine tubes at different stages of the oestrous cycle and to provide compelling evidence that the main spermatozoal storage site in the reproductive tract of the bitch is the distal UTJ.

Proteomic and phenotypic analysis of triclosan tolerant verocytotoxigenic Escherichia coli O157:H19.

Triclosan is a biocidal active agent commonly used in domestic and industrial formulations. Currently, there is limited understanding of the mechanisms involved in triclosan tolerance in Escherichia coli O157. The aim of this study was to identify the differences between a triclosan susceptible E. coli O157:H19 isolate (minimum inhibitory concentration; MIC 6.25 µg/ml) and its triclosan tolerant mutant (MIC>8000 µg/ml) at a proteomic and phenotypic level. Two dimensional DIGE was used to identify differences in protein expression between the reference strain and triclosan tolerant mutant in the presence and absence of triclosan. DIGE analysis indicates the proteome of the reference E. coli O157:H19 was significantly different to its triclosan tolerant mutant. Significant changes in protein expression levels in the triclosan tolerant mutant included the known triclosan target FabI which encodes enoyl reductase, outer membrane proteins and the filament structural protein of flagella, FliC. Phenotypic studies showed that the triclosan tolerant mutant MIC decreased in the presence of efflux inhibitor phenyl-arginine-ß-naphthylamide and biofilm formation was increased in the mutant strain. The data generated indicates that enhanced triclosan tolerance is a result of multiple mechanisms which act together to achieve high-level resistance, rather than mutation of FabI alone.

Perturbed sperm-epithelial interaction in bitches with mating-induced endometritis.

In several species there is a transient uterine inflammatory response after mating that is purported to clear excess and dead spermatozoa, bacteria and other contaminants from the uterus. In particular individuals this inflammatory response is substantial, resulting in an acute mating-induced endometritis, causing infertility. In this study, the influx of polymorphonuclear neutrophils (PMNs) into the uterine lumen of bitches was investigated after artificial insemination with fresh semen. In normal bitches, an influx of PMNs was detected, followed by high pregnancy rates and normal litter size, and may be a physiological inflammatory response. In bitches with endometrial hyperplasia, there was a larger influx of PMNs and pregnancy rates and litter size were reduced, although the effect was partly ameliorated by the post-mating administration of antibiotics. It is postulated that in bitches with endometrial hyperplasia, post-mating endometritis develops with the potential to affect reproduction adversely. In vitro studies demonstrated a reduced ability of spermatozoa to attach to the uterine epithelium of bitches with endometrial hyperplasia. Moreover, PMNs in the co-culture system inhibited spermatozoal attachment to normal and hyperplastic uterine epithelium, especially hyperplastic epithelium. It was concluded that decreased spermatozoal attachment to uterine epithelium mediates a reduction in fertility of bitches with endometrial hyperplasia. This is the first study to detail an apparent physiological uterine inflammatory response to spermatozoa and its perturbation in bitches with endometrial disease, and the first to recognise the clinical significance and potential aetiology of mating-induced endometritis.

The effect of cryopreservation on the capacitation status and epithelial cell attachment capability of dog spermatozoa.

Freezing and cooling of spermatozoa during cryopreservation for artificial insemination causes ultrastructural changes in the acrosome and plasma membrane which reduces longevity and fertility. Cryopreservation-induced capacitation-like changes and reduced ability of spermatozoa to bind to the cells of the reproductive tract of the bitch may contribute to the reduced fertility of cryopreserved spermatozoa. Previous studies in the dog have investigated the effects of extending and cooling spermatozoa on the plasma membrane but often only after freeze-thawing and not in conjunction with an assessment of their ability to bind to uterine tube epithelial explants. This study investigated the effect of each stage of the cryopreservation process on capacitation and attachment to the reproductive tract of the bitch. The capacitation status of spermatozoa was studied over time after cryopreservation using a chlortetracycline and Hoechst 33258 stain. The ability of spermatozoa to bind to uterine tube epithelial explants was studied using Hoechst 33342 stain. Extending, cooling and freeze-thawing promoted capacitation and decreased the spermatozoal binding ability. The effect of each stage appeared to be cumulative with the freeze-thawing stage being the most dramatic. The results suggested that the cumulative effect of each stage of the cryopreservation process results in the promotion of capacitation before spermatozoa have reached the site of fertilisation, and therefore spermatozoa have reduced ability to attach to epithelial cells. These effects are contributory factors to the reduced fertility of cryopreserved spermatozoa.

Application of quantitative reverse-transcription PCR (qRT-PCR) for the determination of the total viable count (TVC) on meat samples.

AIM: To apply a quantitative reverse-transcription PCR (qRT-PCR) method to determine the total viable count (TVC) on meat samples. METHODS AND RESULTS: Using two sets of primers to target the ribonuclease-P (RNase P) RNA transcripts of Gram-positive and Gram-negative bacteria, standard curves were generated using the LightCycler 2.0 instrument (Roche Diagnostics). RNA standards were extracted from known cell numbers and subsequently converted to cDNA for the construction of standard curves for quantification of the TVC of beef carcass swabs (n = 60) and beef (n = 30), chicken (n = 50) and pork (n = 49) pieces. A high correlation between the standard plate count method and the qRT-PCR was observed for beef swabs (R(2) = 0·93) and beef pieces (R(2) = 0·82). The correlation coefficient for chicken pieces and pork pieces were R(2) = 0·34 and 0·55, respectively. Using beef pieces (n = 13), an interlaboratory study was conducted and each participating laboratory (n = 3) found a reasonable degree of agreement between the cultural method and the PCR method. CONCLUSIONS: The qRT-PCR assay used in this study can enumerate the total bacteria on beef samples with a high degree of accuracy. SIGNIFICANCE AND IMPACT OF THE STUDY: The qRT-PCR method may have the potential to be applied to various sample types as an alternative rapid method for determining TVCs; however, further validation would be required.

Treatment of facial asymmetry with poly-L-lactic Acid: a case study.

Poly-L-lactic acid (PLLA) (Sculptra, Dermik Laboratories, Bridgewater, NJ, a business of sanofi-aventis US, LLC) is a novel biocompatible and biodegradable injectable device currently under review by the Food and Drug Administration for a cosmetic indication. When implanted into soft tissues, PLLA is thought to elicit a foreign body reaction, resulting in fibroplasia and subsequent collagen formation. This process leads to a gradual thickening of the dermis and long-lasting augmentation of facial contours. In the reported case, PLLA was used to treat a 69-year-old African-American woman who had undergone numerous unsuccessful procedures for correction of prominent facial asymmetry. One vial of PLLA (5-ml dilution) was injected into the dermal-subcutaneous plane using a lattice distribution in the right upper cheek and malar regions, followed by massage. The procedure was repeated 6 weeks later. Several months after the two separate PLLA treatment sessions, the contours of the right upper cheek and malar regions were visibly and cumulatively enhanced, and facial symmetry was gradually restored, to the patient's full satisfaction. The treatment was well tolerated on both occasions, and the benefits of treatment have been sustained 18 months after the last procedure. The author concludes that injectable PLLA is a safe and effective minimally invasive treatment for facial contour defects.

Relationship between the fertile period and sperm transport in the bitch.

Following ejaculation into the vagina, dog sperm remain functionally competent for many days, acquire the ability to fertilize, and are delivered to an appropriate site within the uterine tube synchronously with the appearance of fertile oocytes. The mechanisms involved in regulating this system are complex, and allow for sperm storage within the female reproductive tract. The aim of this paper is to review current knowledge of the transportation of sperm and their biology within the reproductive tract of the bitch.

Cervical epidural abscess: a rare complication of intravenous cannulation.

Epidural abscess is a rare but life threatening condition that requires early diagnosis and prompt management. We report a case of cervical epidural abscess following an intravenous cannula site infection. The organism responsible was Methicillin Resistant Staphylococcus Aureus (MRSA). Management involved surgery, prolonged antibiotics and ventilation. Despite this, there was no return of neurological function and the patient died. We review the literature on epidural abscesses unrelated to catheterisation or instrumentation of the epidural space and discuss the aetiology and clinical features of this acute medical emergency.

Vital and ultrastructural changes in dog spermatozoa during cyopreservation.

The exact nature of cryo-injury to dog semen and the stage of cryopreservation at which it occurs have not been evaluated fully and were investigated in the present study. Semen samples were examined immediately before and after addition of semen extender, cooling and freeze-thawing in a Tris-based medium. Vital and ultrastructural assessments were made immediately after each treatment and after incubation for 4 h at 39 degrees C after each treatment. Addition of semen extender produced no significant ultrastructural changes in spermatozoa; however, cooling resulted in an immediate increase in the number of acrosomal abnormalities and a subsequent decrease in sperm viability. Freeze-thawing caused both an immediate and a delayed decrease in sperm viability. This effect may have been an exacerbation of the effects of cooling as many acrosomal abnormalities were present or it may have been the result of initially unrecognized damage. Cooling and freezing of dog spermatozoa may have both immediate and delayed effects on the ultrastructure of spermatozoa. The immediate effects of cooling and freezing may either kill spermatozoa or render them incapable of fertilization by damaging the acrosome, whereas delayed effects may reduce sperm longevity by altering plasma membrane structure.

Presence of mature sperm in testicular parenchyma of men with nonobstructive azoospermia: prevalence and predictive factors.

OBJECTIVES: Hitherto, patients with testicular dysfunction and azoospermia had to resort to adoption, donor sperm insemination, or child-free living. The realization that a proportion of such men harbor spermatozoa in their testicular parenchyma, combined with the ability of intracytoplasmic sperm injection (ICSI) to effect pregnancy with single sperm, has prompted male infertility clinicians to explore testicular sperm extraction (TESE) in this patient population. We sought to investigate the likelihood of finding spermatozoa during TESE from men presenting with nonobstructive azoospermia and to define if any factors existed that were predictive of eventual sperm presence or absence. METHODS: Thirty patients with nonobstructive azoospermia underwent TESE and simultaneous formal testis biopsy, cytologic analysis, and wet preparation analysis. Tissue obtained from TESE was analyzed according to a rigorous protocol, followed by exhaustive searching by trained embryologists. RESULTS: Twenty-one patients (70%) had spermatozoa found on testicular tissue analysis. Neither patient age nor follicle-stimulating hormone (FSH) level was predictive of the ability to find sperm. With regard to histologic pattern, 50% of men with Sertoli cell-only, 75% of patients with maturation arrest, and 100% of patients with spermatids seen on histologic analysis had sperm retrieved from their testicular tissue during TESE. Absence of sperm on cytologic smear and wet preparation analysis failed to predict the presence of sperm on formal testicular tissue analysis in 40% of patients. CONCLUSIONS: Men with nonobstructive azoospermia may have mature spermatozoa present within their testicular parenchyma. Relying on these data, patients should not be excluded from TESE based on serum FSH level, age, prior histopathologic pattern, or cytology/wet preparation results. These figures will allow clinicians to counsel patients with nonobstructive azoospermia informatively regarding TESE and their chances of having testicular sperm retrieved.

Efficacy of intracytoplasmic sperm injection using intentionally cryopreserved epididymal spermatozoa.

Microsurgical epididymal sperm aspiration was a great advance in the therapy of patients with non-reconstructable, obstructive azoospermia, most notably congenital bilateral absence of the vas deferens. Using conventional in-vitro fertilization, pregnancies were rarely achieved because the rate of oocyte fertilization was extremely poor. However, the use of retrieved spermatozoa in conjunction with intracytoplasmic sperm injection (ICSI) has dramatically increased the likelihood of embryo formation. Typically, sperm and oocyte harvesting are performed simultaneously. We have investigated whether frozen-thawed spermatozoa work as well as fresh spermatozoa. When we had concluded from our own population of patients (groups I and II) that they did, we adopted a policy of aspirating spermatozoa, primarily cryopreserving them and using them for ICSI at a later date. We found the fertilization rates of this latter cohort of patients (group III) to be excellent (37% per oocyte), and the ongoing pregnancy rate is quite satisfactory (40% per couple, 29% per cycle). We offer this approach as an alternative to the traditional scheme because it markedly eases the burden of partner scheduling on both the couple and the clinicians involved. In addition, assurance of the availability of male partner spermatozoa can be attained prior to beginning ovulation induction.

The surgical treatment of patent ductus arteriosus.1955.

Fourteen cases of patent ductus arteriosus operated upon without a fatality have been presented. All were treated by ligation and division of the ductus. The importance of the safety factor provided by the Potts-Smith-Gibson clamp has been discussed. A detailed case report of one patient whose operation was complicated by excessive hemorrhage is given in detail. No cerebral or renal damage followed a prolonged period of cardiac massage and only slight permanent cord damage resulted from complete occlusion of the aorta for over 1 hour.