Enterococcus species distribution among human and animal hosts using multiplex PCR

Aims: This study evaluated the use of Enterococcus species differentiation as a tool for microbial source tracking (MST) in recreational waters. Methods and Results: Avian, mammalian and human faecal samples were screened for the occurrence of Enterococcus avium, Enterococcus casseliflavus, Enterococcus durans, Enterococcus gallinarum, Enterococcus faecium, Enterococcus faecalis, Enterococcus hirae and Enterococcus saccharolyticus using multiplex PCR. Host‐specific patterns of Enterococcus species presence were observed only when data for multiple Enterococcus species were considered in aggregate. Conclusions: The results suggest that no single Enterococcus species is a reliable indicator of the host faecal source. However, Enterococcus species composite ‘fingerprints’ may offer auxiliary evidence for bacterial source identification. Significance and Impact of Study: This study presents novel information on the enterococci species assemblages present in avian and mammalian hosts proximate to the nearshore ocean. These data will aid the development of appropriate MST strategies, and the approach used in this study could potentially assist in the identification of faecal pollution sources.     

Identification of a globally distributed clinical streptomycin-resistance plasmid and other resistance determinants in a coastal bay of China

Aims: To study streptomycin‐resistant bacteria isolated from Jiaozhou Bay and their molecular determinants of resistance. Methods and Results: Twenty‐seven tetracycline‐resistant and 49 chloramphenicol‐resistant bacterial isolates from surface seawater of Jiaozhou Bay were selected for investigation. More than 88% of these isolates were resistant to streptomycin. Half of the streptomycin‐resistant bacteria harboured the strA–strB gene pair, and six isolates carried Tn5393‐like transposons by PCR detection. The p9123‐related plasmids containing the sul2–strA–strB gene cluster were characterized in two environmental Escherichia coli isolates. Transposon Tn5393 was first identified on a Klebsiella pneumoniae plasmid, which also carried Tn1721, estP and umu genes responsible for antimicrobial and insecticide resistance. Conclusions: Coresistance to streptomycin and tetracycline or chloramphenicol was found with high frequency. p9123‐related plasmid and Tn5393 transposon may contribute to the wide distribution and spread of the strA–strB gene pair in Jiaozhou Bay. The detection of streptomycin‐resistance plasmid pQ1‐1 from Jiaozhou Bay seawater bacteria and human bacterial pathogens from USA indicates its global dissemination and transmission, across different components of the microbiota on earth. Significance and Impact of the Study: Streptomycin resistance can be recognized as an important bioindicator of environmental quality, owing to its association with anthropogenic pollution and the multidrug‐resistant microbiota.     

Covalently linked immunomagnetic separation/adenosine triphosphate technique (Cov‐IMS/ATP) enables rapid,in‐field detection and quantification of Escherichia coli and Enterococcus spp. in freshwater and marine environments

Aims: Developing a rapid method for detection of faecal pollution is among the critical goals set forth by the Environmental Protection Agency in its revision of water quality criteria. The purpose of this study is to devise and test covalently linked antibody–bead complexes for faecal indicator bacteria (FIB), specifically Escherichia coli or Enterococcus spp., in measuring water quality in freshwater and marine systems. Methods and Results: Covalently linked complexes were 58–89% more robust than antibody–bead complexes used in previous studies. Freshwater and marine water samples analysed using covalently linked immunomagnetic separation/adenosine triphosphate quantification technique (Cov‐IMS/ATP) and culture‐based methods yielded good correlations for E. coli (R = 0·87) and Enterococcus spp. (R = 0·94), with method detection limits below EPA recreational water quality health standards for single standard exceedances (E. coli– 38 cells per 100 ml; Enterococcus spp. – 25 cells per 100 ml). Cov‐IMS/ATP correctly classified 87% of E. coli and 94% of Enterococcus spp. samples based on these water quality standards. Cov‐IMS/ATP was also used as a field method to rapidly distinguish differential loading of E. coli between two stream channels to their confluence. Conclusions: Cov‐IMS/ATP is a robust, in‐field detection method for determining water quality of both fresh and marine water systems as well as differential loading of FIB from two converging channels. Significance and Impact of the Study: To our knowledge, this is the first work to present a viable rapid, in‐field assay for measuring FIB concentrations in marine water environments. Cov‐IMS/ATP is a potential alternative detection method, particularly in areas with limited laboratory support and resources, because of its increased economy and portability.     

Effect of biofilm in irrigation pipes on microbial quality of irrigation water

Aims: The focus of this work was to investigate the contribution of native Escherichia coli to the microbial quality of irrigation water and to determine the potential for contamination by E. coli associated with heterotrophic biofilms in pipe‐based irrigation water delivery systems. Methods and Results: The aluminium pipes in the sprinkler irrigation system were outfitted with coupons that were extracted before each of the 2‐h long irrigations carried out with weekly intervals. Water from the creek water and sprinklers, residual water from the previous irrigation and biofilms on the coupons were analysed for E. coli. High E. coli concentrations in water remaining in irrigation pipes between irrigation events were indicative of E. coli growth. In two of the four irrigations, the probability of the sample source, (creek vs sprinkler), being a noninfluential factor, was only 0·14, that is, source was an important factor. The population of bacteria associated with the biofilm on pipe walls was estimated to be larger than that in water in pipes in the first three irrigation events and comparable to one in the fourth event. Conclusion: Biofilm‐associated E. coli can affect microbial quality of irrigation water and, therefore, should not be neglected when estimating bacterial mass balances for irrigation systems. Significance and Impact of the Study: This work is the first peer‐reviewed report on the impact of biofilms on microbial quality of irrigation waters. Flushing of the irrigation system may be a useful management practice to decrease the risk of microbial contamination of produce. Because microbial water quality can be substantially modified while water is transported in an irrigation system, it becomes imperative to monitor water quality at fields, rather than just at the intake.     

Efficacy of multiple metals against copper-resistant bacterial strains

Aims: The antibacterial efficacy of zeolites containing copper (Cu) or silver (Ag) ions or a combination was assessed against several reported copper‐resistant (Cu^R) bacterial strains. Methods and Results: Comparison strains were obtained from the American Type Culture Collection that had no documented metal resistance. Reductions in bacterial populations were determined after exposure time intervals of 3, 6 and 24 h. All three Cu^R strains of Salmonella enterica exhibited resistance to Cu, Ag and Cu/Ag after three and 6 h of exposure. Both the Cu^R and comparison strain of Enterococcus faecium were resistant to both metals and the metal combination. Cu^RPseudomonas putida was significantly reduced by all zeolites within 3 h. The Cu^REscherichia coli strain was more sensitive to Cu, but more resistant to Ag than the comparison strain; however, significant reductions were achieved within 3 h with both Cu and Cu/Ag, and within 24 h with Ag. Conclusions: Some strains with reported resistance to Cu were also resistant to Ag, suggestive of a shared resistance mechanism such as an indiscriminate Cu efflux pump. Ent. faecium appears to have innate resistance to both metals. In general, Ent. faecium was the most resistant species to the individual metals and the combination of metals, Ps. putida the least resistant, and the Salmonella strains were more resistant than E. coli. Significance and Impact of the Study: Several of the comparison strains with no reported copper resistance were resistant to one or both metals. This may call into question the methods for determining bacterial metal resistance, which typically use nutrient‐rich media containing metals to assess the ability of the bacteria to grow in comparison with a wild‐type strain. Nevertheless, all the Cu^R strains evaluated in this study, with the exception of Ent. faecium, were reduced using the Cu and Ag zeolite combination.     

Automated dead-end ultrafiltration of large volume water samples to enable detection of low-level targets and reduce sample variability

Aims

A Portable Multi‐use Automated Concentration System (PMACS) concentrates micro‐organisms from large volumes of water through automated dead‐end ultrafiltration and backflushing. The ability to detect microbial targets from ground, surface and cooling tower waters collected using standard methods was compared with samples from the PMACS in this study.

Methods and Results

PMACS (100 l) and standard grab samples (100–500 ml) were collected from sites in Florida and South Carolina, USA. Samples were analysed for the presence of faecal indicator bacteria (FIB; ground and surface water) or Legionella pneumophila (Lp; cooling tower water). FIB were enumerated by growth on selective media following membrane filtration or in IDEXX defined substrate media. Lp cells were detected by direct fluorescence immunoassay using FITC‐labelled monoclonal antibodies targeting serogroups 1, 2, 4 and 6. FIB were found in PMACS samples from ground and surface waters when their concentrations were below detection limits in grab samples. The concentrations of Lp in cooling tower samples collected over 5 months were more consistent in PMACS samples than grab samples.

Conclusions

These data demonstrate that PMACS concentration is advantageous for water monitoring. FIB were detected in PMACS samples when their concentrations were below the detection limits of the standard methods used. PMACS processing provided more representative samples of cooling tower waters reducing sample variability during long‐term monitoring.

Significance and Impact of the Study

This study highlights the utility of PMACS processing for enhanced monitoring of water for low‐level microbial targets and for reducing sample variability in long‐term monitoring programmes.     

Indicator microbes correlate with pathogenic bacteria, yeasts and helminthes in sand at a subtropical recreational beach site

Aims: Research into the relationship between pathogens, faecal indicator microbes and environmental factors in beach sand has been limited, yet vital to the understanding of the microbial relationship between sand and the water column and to the improvement of criteria for better human health protection at beaches. The objectives of this study were to evaluate the presence and distribution of pathogens in various zones of beach sand (subtidal, intertidal and supratidal) and to assess their relationship with environmental parameters and indicator microbes at a non‐point source subtropical marine beach. Methods and Results: In this exploratory study in subtropical Miami (Florida, USA), beach sand samples were collected and analysed over the course of 6 days for several pathogens, microbial source tracking markers and indicator microbes. An inverse correlation between moisture content and most indicator microbes was found. Significant associations were identified between some indicator microbes and pathogens (such as nematode larvae and yeasts in the genus Candida), which are from classes of microbes that are rarely evaluated in the context of recreational beach use. Conclusions: Results indicate that indicator microbes may predict the presence of some of the pathogens, in particular helminthes, yeasts and the bacterial pathogen Staphylococcus aureus including methicillin‐resistant forms. Indicator microbes may thus be useful for monitoring beach sand and water quality at non‐point source beaches. Significance and Impact of the Study: The presence of both indicator microbes and pathogens in beach sand provides one possible explanation for human health effects reported at non‐point sources beaches.     

Presence of Giardia cysts and Cryptosporidium oocysts in drinking water supplies in northern Spain

AIMS: To evaluate the prevalence of Cryptosporidium and Giardia in surface water supplies from the province of Alava, northern Spain, and to investigate possible associations among the presence of these pathogenic protozoa with microbiological, physicochemical and atmospheric parameters. METHODS AND RESULTS: A total of 284 samples of drinking and recreational water supplies were analysed. Cryptosporidium oocysts were found in 63.5% of river samples, 33.3% of reservoirs samples, 15.4% and 22.6% of raw water samples from conventional and small water treatment facilities (respectively), 30.8% of treated water from small treatment facilities, and 26.8% of tap water from municipalities with chlorination treatment only. Giardia cysts were found in 92.3% of river samples, 55.5% of reservoirs samples, 26.9% and 45.2% of raw water samples from conventional and small water treatment facilities (respectively), 19.2% of treated water from small treatment facilities, and 26.8% of tap water from municipalities with chlorination treatment only. The presence of Cryptosporidium and Giardia had significant Pearson's correlation coefficients (P < 0.01) with the turbidity levels of the samples, and a number of significant associations were also found with the count levels for total coliforms and Escherichia coli. The samples were positive for Cryptosporidium significantly (P < 0.05) more frequently during the autumn season than during the spring and winter seasons. No significant differences were found in the seasonal pattern of Giardia. A moderate association (r = 0.52) was found between rainfall and the presence of Cryptosporidium oocysts. CONCLUSIONS: Cryptosporidium and Giardia are consistently found at elevated concentrations in surface waters for human consumption from the province of Alava, northern Spain. SIGNIFICANCE AND IMPACT OF THE STUDY: Water treatments based on rapid filtration process and/or chlorination only are often unsatisfactory to provide safe drinking water, a situation that represents an important public health problem for the affected population because of the risk of waterborne outbreaks.     

Hidden reservoirs of pathogens in dental settings

Nosocomial infections are a major concern to both clinicians and health care seekers. Investigations have suggested that laptops & mobile phones may contribute to cross-contamination and can serve as vehicles for infection transmission. Therefore, it is of interest to document the data on hidden reservoirs such as mobile phones and laptops of pathogens in dental settings at the Hazaribag College of dental sciences and Hospital, Jharkhand. The samples were collected from 25 laptops and 25 mobile phones from dentists working in a dental college in Hazaribag city. The samples were collected aseptically using sterile cotton swabs dipped in sterile saline by rotating the swabs on the keyboard surfaces of laptops and mobile phones, inoculated into Brain Heart Infusion broth, vortexed for 1 minute in Fischer Vortex Genie 2 on highest setting & streaked immediately on 5% sheep blood agar plates and were incubated at 37°C for 24 hours aerobically. The isolates were identified based on the colony morphology, colony characteristics and biochemical reactions. The bacterial species isolated were Staphylococcus aureus, Coagulase negative Staphylococcus, Bacillus species, Enterococci, Micrococci, and Pseudomonas etc. Predominant species isolated was Staphylococcus aureus and least was Micrococci. Higher percentage of organisms was found at the Department of Periodontics, Endodontics and least was found in Department of Public Health Dentistry. The percentage and type of organism isolated from keyboards of laptops and mobile phones were similar. Thus, laptops and mobile phones act as vehicles for transfer of potential pathogens associated with dental hospitals. Disinfecting the hands prior to examination of patients and disinfection of laptops and mobiles with alcohol wipes should be done to prevent nosocomial infections.     

A rapid and simple PCR method for identifying isolates of the genus Azospirillum within populations of rhizosphere bacteria

Aims: Escherichia coli is the pre‐eminent microbiological indicator used to assess safety of drinking water globally. The cost and equipment requirements for processing samples by standard methods may limit the scale of water quality testing in technologically less developed countries and other resource‐limited settings, however. We evaluate here the use of ambient‐temperature incubation in detection of E. coli in drinking water samples as a potential cost‐saving and convenience measure with applications in regions with high (>25°C) mean ambient temperatures. Methods and Results: This study includes data from three separate water quality assessments: two in Cambodia and one in the Dominican Republic. Field samples of household drinking water were processed in duplicate by membrane filtration (Cambodia), Petrifilm? (Cambodia) or Colilert^® (Dominican Republic) on selective media at both standard incubation temperature (35–37°C) and ambient temperature, using up to three dilutions and three replicates at each dilution. Matched sample sets were well correlated with 80% of samples (n = 1037) within risk‐based microbial count strata (E. coli CFU 100 ml^?1 counts of <1, 1–10, 11–100, 101–1000, >1000), and a pooled coefficient of variation of 17% (95% CI 15–20%) for paired sample sets across all methods. Conclusions: These results suggest that ambient‐temperature incubation of E. coli in at least some settings may yield sufficiently robust data for water safety monitoring where laboratory or incubator access is limited. Significance and Impact of the Study: Ambient‐temperature incubation of E. coli may be a promising option for reducing the complexity and costs associated with water safety monitoring for faecal indicator bacteria such as E. coli in a field context in resource‐limited settings, as are often encountered in developing countries and after disasters.     

Glass wool filters for concentrating waterborne viruses and agricultural zoonotic pathogens

The key first step in evaluating pathogen levels in suspected contaminated water is concentration. Concentration methods tend to be specific for a particular pathogen group, for example US Environmental Protection Agency Method 1623 for Giardia and Cryptosporidium, which means multiple methods are required if the sampling program is targeting more than one pathogen group. Another drawback of current methods is the equipment can be complicated and expensive, for example the VIRADEL method with the 1MDS cartridge filter for concentrating viruses. In this article we describe how to construct glass wool filters for concentrating waterborne pathogens. After filter elution, the concentrate is amenable to a second concentration step, such as centrifugation, followed by pathogen detection and enumeration by cultural or molecular methods. The filters have several advantages. Construction is easy and the filters can be built to any size for meeting specific sampling requirements. The filter parts are inexpensive, making it possible to collect a large number of samples without severely impacting a project budget. Large sample volumes (100s to 1,000s L) can be concentrated depending on the rate of clogging from sample turbidity. The filters are highly portable and with minimal equipment, such as a pump and flow meter, they can be implemented in the field for sampling finished drinking water, surface water, groundwater, and agricultural runoff. Lastly, glass wool filtration is effective for concentrating a variety of pathogen types so only one method is necessary. Here we report on filter effectiveness in concentrating waterborne human enterovirus, Salmonella enterica, Cryptosporidium parvum, and avian influenza virus.