Could honey bees signal the spread of antimicrobial resistance in the environment?

The honey bee has long been known to be a bioindicator of environmental pollution and the use of antimicrobials in the beekeeping industry is strictly regulated. For these reasons, this paper was aimed to evaluate for the first time the role of Apis mellifera as a possible indicator of environmental antimicrobial resistance (AMR). The study isolated and analysed the resistance patterns of Enterobacteriaceae from a pool of honey bee guts located in five different environmental sites (ES), where different antimicrobial selective pressures were hypothesized. In all, 48 isolates were considered for identification and underwent analyses of AMR to ampicillin, amoxicillin/clavulanic acid, cefazolin, ceftazidime, tetracycline, imipenem, enrofloxacin, amikacin and trimethoprim/sulfamethoxazole. In all, 12 isolates out of 48 (25%) showed resistance to at least one antimicrobial drug. There were no significant differences between the resistance rates observed in the ESs, even if the highest percentage of resistance was found in ES4. Resistances to amoxicillin/clavulanic acid resulted significantly higher than those detected towards the other antimicrobials. Amoxicillin/clavulanic acid is not commonly used in beekeeping but it is extensively used in animals and in humans, suggesting an environmental origin of this resistance and supporting the hypothesis that honey bees could be used as indicators of AMR spread in the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, a possible role of honey bees as indicator of environmental antimicrobial resistance is hypothesized. Enterobacteriaceae were isolated from bees living in different environmental sites (ES) where different antimicrobial selective pressures were hypothesized. Even if no differences between the resistances in the five ES were observed, the resistance rates for amoxicillin/clavulanic acid, compared to other antimicrobials, were significantly higher. Since amoxicillin/clavulanic acid is not used in beekeeping but it is extensively used in animals and in humans, an environmental origin of this resistance is suggested that supports our hypothesis.

First description of Streptococcus lutetiensis from a diseased cat.

This paper describes for the first time the isolation of Streptococcus lutetiensis in a cat with intestinal lymphoma. The Streptococcus bovis group has undergone significant taxonomic changes over the past two decades and, in 2002, Poyart et al. described two distinct novel species within the genus Streptococcus: Streptococcus lutetiensis and Streptococcus pasteurianus. The bovis group streptococci include commensal species and subspecies or opportunistic pathogens of humans and animals. The cat was referred to the Veterinary Teaching Hospital, University of Bologna for chronic diarrhoea associated with fresh blood. A diagnosis of intestinal lymphoma was advanced. S. lutetiensis was accidentally isolated from the faeces of the cat and identified through MALDI-TOF and 16s rRNA sequencing. The Kirby-Bauer test revealed that the isolate was resistant to enrofloxacin, erythromycin, clindamycin, marbofloxacin and tetracycline. The detection of S. lutetiensis in cat faeces might suggest that it could be a normal inhabitant of cat intestinal tract or that it could be involved in the manifestation of intestinal diseases. Since bacteria belonging to the S. bovis group are considered emerging pathogens, additional research is required to evaluate the role of S. lutetiensis in cats and its role in the transmission of antimicrobial resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study the isolation of Streptococcus lutetiensis from a cat with intestinal lymphoma was described for the first time. An antimicrobial susceptibility test performed by means of the disc diffusion method revealed that the isolate was resistant to enrofloxacin, erythromycin, clindamycin, marbofloxacin and tetracycline. Nowadays the ecological or pathogenetic role of S. lutetiensis in the gut of animals remains unclear but, even if its role as commensal bacterium was confirmed, the presence of multi-resistant S. lutetiensis in cat gut could favour the transmission of antimicrobial resistance to other bacteria.

Occurrence of hepatitis A and E and norovirus GI and GII in ready-to-eat vegetables in Italy.

Fresh vegetables and their ready-to-eat (RTE) salads have become increasingly recognized as potential vehicles for foodborne diseases. The EU Reg. 1441/2007 establishes microbiological criteria for bacterial pathogens for products placed on the market during their shelf-life (i.e. Salmonella spp., Listeria monocytogenes) for pre-cut fruits and vegetables (RTE) whilst it does not address the problem of contamination by enteric viruses. In this study we investigated the contamination by hepatitis A virus (HAV), hepatitis E virus (HEV) and norovirus (NoV) in 911 ready-to-eat vegetable samples taken from products at retail in Apulia and in Lombardia. The vegetable samples were tested using validated real-time PCR (RT-qPCR) assays, ISO standardized virological methods and ISO culturing methods for bacteriological analysis. The total prevalence of HAV and HEV was 1.9% (18/911) and 0.6% (6/911), respectively. None of the samples analysed in this study was positive for NoV, Salmonella spp. or Listeria monocytogenes. The detection of HAV and HEV in RTE salads highlights a risk to consumers and the need to improve production hygiene. Appropriate implementation of hygiene procedures is required at all the steps of the RTE vegetable production chain and this should include monitoring of emerging viral pathogens.

Occurrence of putative virulence genes on Arcobacter butzleri isolated from three different environmental sites throughout the dairy chain.

AIMS: This comparative study investigated the occurrence of cadF, cj1349, ciaB, pldA, tlyA, hecA, hecB, mviN, irgA and IroE genes in 212 Arcobacter butzleri isolated from three different environmental sites linked to the dairy chain (farms, industrial and artisanal dairy plants) located in three Italian regions (Lombardy, Emilia-Romagna and Calabria). METHODS AND RESULTS: According to the presence of these genes, different pathotypes (P-types) were determined. The main genes detected were ciaB, mviN, tlyA, cj1349, pldA and cadF, while the least common genes were iroE, hecA, hecB and irgA. TlyA, irgA, hecA, hecB and iroE, which were significantly more frequent in isolates recovered in industrial dairy plants. Twelve P-types were detected. The occurrence of the most frequently detected P-types (P-types 1, 2, 3 and 5) differed significantly (P < 0·001) in relation to both the environmental site and geographical area of isolation. The highest diversity in P-types was observed in industrial dairy plants and in the Calabria region. CONCLUSIONS: The results of this study show a correlation between the occurrence of putative virulence genes and virulence genotype variability depending on the environmental site and geographical origin of the isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides insights into the similar distribution of putative virulence genes in a dairy chain and other sources' isolates and also into a geographical distribution of some P-types. We have shown that industrial dairy plants may represent an environmental site favouring a selection of the isolates with a higher pathogenetic pattern.

Paediatric HUS Cases Related to the Consumption of Raw Milk Sold by Vending Machines in Italy: Quantitative Risk Assessment Based on Escherichia coli O157 Official Controls over 7 years.

A quantitative risk assessment (RA) was developed to estimate haemolytic-uremic syndrome (HUS) cases in paediatric population associated with the consumption of raw milk sold in vending machines in Italy. The historical national evolution of raw milk consumption phenomenon since 2008, when consumer interest started to grow, and after 7 years of marketing adjustment, is outlined. Exposure assessment was based on the official Shiga toxin-producing Escherichia coli O157:H7 (STEC) microbiological records of raw milk samples from vending machines monitored by the regional Veterinary Authorities from 2008 to 2014, microbial growth during storage, consumption frequency of raw milk, serving size, consumption preference and age of consumers. The differential risk considered milk handled under regulation conditions (4°C throughout all phases) and the worst time-temperature field handling conditions detected. In case of boiling milk before consumption, we assumed that the risk of HUS is fixed at zero. The model estimates clearly show that the public health significance of HUS cases due to raw milk STEC contamination depends on the current variability surrounding the risk profile of the food and the consumer behaviour has more impact than milk storage scenario. The estimated HUS cases predicted by our model are roughly in line with the effective STEC O157-associated HUS cases notified in Italy only when the proportion of consumers not boiling milk before consumption is assumed to be 1%. Raw milk consumption remains a source of E. coli O157:H7 for humans, but its overall relevance is likely to have subsided and significant caution should be exerted for temporal, geographical and consumers behaviour analysis. Health education programmes and regulatory actions are required to educate people, primarily children, on other STEC sources.

Short communication: Investigation into Mycobacterium avium ssp. paratuberculosis in pasteurized milk in Italy.

This study investigated the presence of viable Mycobacterium avium ssp. paratuberculosis (MAP) in pasteurized milk produced by Italian industrial dairy plants to verify the prediction of a previously performed risk assessment. The study analyzed 160 one-liter bottles of pasteurized milk from 2 dairy plants located in 2 different regions. Traditional cultural protocols were applied to 500mL of pasteurized milk for each sample. The investigation focused also on the pasteurization parameters and data on the microbiological characteristics of raw milk (total bacterial count) and pasteurized milk (Enterobacteriaceae and Listeria monocytogenes). No sample was positive for MAP, the pasteurization parameters complied with European Union legislation, and the microbiological analysis of raw and pasteurized milk showed good microbiological quality. The results show that a 7-log (or >7) reduction could be a plausible value for commercial pasteurization. The combination of hygiene practices at farm level and commercial pasteurization yield very low or absent levels of MAP contamination in pasteurized milk, suggesting that pasteurized milk is not a significant source of human exposure to MAP in the dairies investigated.

Multilocus sequence typing of Arcobacter butzleri isolates collected from dairy plants and their products, and comparison with their PFGE types.

AIMS: The present study aimed to determine, by multilocus sequence type (MLST), the heterogeneity level of Arcobacter butzleri isolates and to compare MLST and pulsed-field gel electrophoresis (PFGE) in terms of discriminatory power (DI) as well as unidirectional and bi-directional concordance. METHODS AND RESULTS: Arcobacter butzleri isolates (N = 133) from dairy products and environmental samples, collected from dairy plants, were characterized by MLST and PFGE with SacII and classified in 29 sequence types (STs), 47 PFGE and 62 type strains (TS). Among the 119 alleles, 19 were previously unreported and the same for all the STs but two. A significant linkage disequilibrium was detected when the complete ST data set was analysed The DIs of MLST, PFGE and their combination were 0·937, 0·953 and 0·965 respectively. The adjusted Wallace coefficients between MLST and PFGE as well as PFGE and MLST were 0·535 and 0·720 respectively; the adjusted Rand coefficient was 0·612. CONCLUSIONS: The A. butzleri studied population showed recombination to some degree. PFGE showed a DI higher than MLST. Both methods presented good concordance. The TS analysis seems to show persistence of the same strain on time and possible cross-contaminations between food and environmental sites. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides insights in the A. butzleri population found in raw milk, cheese, and dairy production plants. The data suggest that MLST and PFGE genotypes correlate reasonably well, although their combination results in optimal resolution.

Short communication: Arcobacter butzleri and Arcobacter cryaerophilus survival and growth in artisanal and industrial ricotta cheese.

Ricotta cheese is a ready-to-eat product with properties (pH >6.0, aw >0.98-0.99) and moisture content (75-80%) that may pose a risk to public health due to postprocess contamination by several bacterial pathogens, including Arcobacters. The objective of the study was to evaluate the behavior of Arcobacter butzleri and Arcobacter cryaerophilus in ricotta cheese during its shelf life assuming postprocessing contamination. Two types of ricotta cheese, artisanal water buffalo (WB) and industrial cow milk ricotta cheese, were experimentally contaminated with A. butzleri and A. cryaerophilus and the count was monitored at 2 different temperatures (6°C and 12°C) during shelf life of 5 d for WB cheese and 22 d for industrial ricotta cheese. In WB ricotta cheese the A. butzleri count remained stable during the 5 d of storage at 6°C, whereas a moderate but significant decrease was observed in A. cryaerophilus count. The counts of both species increased when WB ricotta cheese was stored at 12°C. In industrial ricotta cheese stored at 6°C, a significant reduction was observed both in A. butzleri and A. cryaerophilus counts during the 22-d storage period; at 12°C storage, a count increase was observed for both Arcobacter species up to d 14 of storage after which the log cfu/g count resulted constant until d 22 of storage. The ability of A. butzleri and A. cryaerophilus to survive at 6°C and to grow at 12°C in ricotta cheese has significant food safety implications.

A screening sampling plan to detect Mycobacterium avium subspecies paratuberculosis-positive dairy herds.

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic contagious bacterial disease primarily affecting dairy cattle. Paratuberculosis represents a dual problem for the milk production chain: in addition to economic losses to affected herds, MAP may have zoonotic potential. Infected herds must be identified in order to implement programs designed to reduce the incidence of disease within and between herds and to prevent MAP from entering the food chain. The objective of this study was to evaluate the sensitivity and specificity of a screening sampling plan (SSP) to detect MAP-positive dairy herds by repetitive analysis of bulk tank milk (BTM) samples by ELISA and in-line milk filter (ILMF) samples by PCR. Samples from BTM and ILMF were collected twice from 569 dairy herds in southern Italy. Additionally, 12,016 individual milk samples were collected: 9,509 from 102 SSP-positive herds (SSP MAP-positive) and 2,507 from 21 randomly selected SSP-negative herds (SSP MAP-negative). There was a total of 126 SSP MAP-positive herds (i.e., 21.3% SSP MAP-positive herds; 95% confidence interval=18.0-24.9); the within-herd apparent prevalence (AP) ranged between 0.00 and 22.73% (mean 6.07%). A significant difference in within-herd AP was shown between SSP MAP-positive herds and SSP MAP-negative herds. A highly significant association was shown between the median AP herd status (>5%) and positivity to at least one ILMF or BTM sample. The SSP detected a minimum of 56.25% of low AP herds (AP ≤ 2.0%) up to a maximum of 100% of herds with a within-herd AP ≥ 8.0%. Overall, the SSP detected 85.57% of herds in which at least one individual milk sample was positive by ELISA. The proposed SSP was an inexpensive and useful tool to detect MAP-positive herds with a higher risk of infection diffusion and milk contamination. Although the SSP cannot be used for MAP-free certification of herds, it could be useful to prioritize appropriate control measures aimed at reducing the prevalence of infection in dairy herds and milk contamination.

Short communication: occurrence of Arcobacter species in industrial dairy plants.

The present study investigated the presence of Arcobacter spp. in industrial dairy plants. Between February and September 2013, pasteurized milk used for cheesemaking, processing and cleaning water, cheese, and environmental samples from different plant sites, including surfaces in contact or not in contact with food, were sampled. A total of 126 samples were analyzed by the cultural method and isolates were identified by multiplex PCR. Arcobacter spp. were isolated from 22 of 75 environmental samples (29.3%): of them, 22.7% were surfaces in contact with food and 38.7% surfaces not in contact with food. A total of 135 Arcobacter spp. isolates were obtained; of these, 129 and 6 were identified as Arcobacter butzleri and Arcobacter cryaerophilus, respectively. All food processing water and pasteurized milk samples were negative for Arcobacter species. We were not able to determine the primary source of contamination, but the isolation of both A. butzleri and A. cryaerophilus in surfaces in contact with food before and during manufacturing suggests that Arcobacter spp. are not or are only partially affected by routine sanitizing procedures in the industrial dairy plants studied. The efficacy of sanitizing procedures should be evaluated and further studies are needed to determine whether certain Arcobacter strains persist for long periods of time in industrial dairy plants and whether they can survive in different types of cheese in cases of postprocessing contamination.

Presence of Campylobacter and Arcobacter species in in-line milk filters of farms authorized to produce and sell raw milk and of a water buffalo dairy farm in Italy.

The objectives of this study were to investigate the presence of Campylobacter spp. and Arcobacter spp. in dairy herds authorized for the production and sale of raw milk and in a water buffalo dairy farm, and to test the antimicrobial susceptibility of the isolates. A total of 196 in-line milk filters were collected from 14 dairy farms (13 bovine and 1 water buffalo) for detection of Campylobacter spp. and Arcobacter spp. by microbiological culture. For each farm investigated, 1 isolate for each Campylobacter and Arcobacter species isolated was tested using the Etest method (AB Biodisk, Solna, Sweden) to evaluate the susceptibility to ciprofloxacin, tetracycline, chloramphenicol, ampicillin, erythromycin, and gentamicin. A total of 52 isolates were detected in 49 milk filters in 12 farms (85.7%) out of 14 and the isolates were identified as Campylobacter jejuni (6), Campylobacter hyointestinalis ssp. hyointestinalis (8), Campylobacter concisus (1), Campylobacter fetus ssp. fetus (1), Arcobacter butzleri (22), and Arcobacter cryaerophilus (14). The small number of isolates tested for antimicrobial susceptibility precludes any epidemiological consideration but highlights that all Campylobacter isolates were susceptible to macrolides, which are the first-choice drugs for the treatment of campylobacteriosis, and that resistance to fluoroquinolones and tetracycline was detected; for Arcobacter isolates, resistance to ampicillin and chloramphenicol was detected. The sale of raw milk for human consumption by self-service automatic vending machines has been allowed in Italy since 2004 and the presence of C. jejuni in in-line milk filters confirms that raw milk consumption is a significant risk factor for human infection. The high occurrence of emerging Campylobacter spp. and Arcobacter spp. discovered in dairy farms authorized for production and sale of raw milk represents an emerging hazard for human health.

Quantitative risk assessment of verocytotoxin-producing Escherichia coli O157 and Campylobacter jejuni related to consumption of raw milk in a province in Northern Italy.

A quantitative risk assessment was developed to describe the risk of campylobacteriosis and hemolytic uremic syndrome (HUS) linked to consumption of raw milk sold in vending machines in Northern Italy. Exposure assessment considered the microbiological status of dairy farms, expected milk contamination, storage conditions from bulk tank to home storage, microbial growth during storage, destruction experiments, consumption frequency of raw milk, age of consumers, serving size, and consumption preference. The differential risk between milk handled under regulation conditions (4°C throughout all phases) and the worst field handling conditions was considered. The probability of Campylobacter jejuni infection was modeled with a single-hit dose-response beta-Poisson model, whereas for HUS an exponential dose-response model was chosen and two probabilities were used to model the higher susceptibility of children younger than 5 years old. For every 10,000 to 20,000 consumers each year, the models predicted for the best and worst storage conditions, respectively, 2.12 and 1.14 campylobacteriosis cases and 0.02 and 0.09 HUS cases in the 0- to 5-year age group and 0.1 and 0.5 HUS cases in the >5-year age group. The expected pediatric HUS cases do not differ considerably from those reported in Italy by the Minister of Health. The model developed may be a useful tool for extending the assessment of the risk of campylobacteriosis and HUS due to raw milk consumption at the national level in Italy. Considering the epidemiological implications of this study, the risk of illness linked to raw milk consumption should not be ignored and could be reduced by the use of simple measures. Boiling milk before consumption and strict control of temperatures by farmers during raw milk distribution have significant effects on campylobacteriosis and HUS and are essential measures for risk management.

Behaviour of Listeria monocytogenes in packaged water buffalo mozzarella cheese.

AIMS: The aim of the study was to evaluate the behaviour of Listeria monocytogenes in the conditioning liquid of packaged water buffalo mozzarella cheese (WBMC). METHODS AND RESULTS: The conditioning liquid was contaminated with L. monocytogenes, and the contaminated samples were stored at four different storage temperatures: 5 and 10 °C for 22 days; 20 °C for 9 days; 20 °C for 3 days and then at 5 °C for 6 days. The results showed that L. monocytogenes concentration decreased when contaminated samples were stored at 5 °C. When WBMC was stored at 20 °C and at 10 °C, L. monocytogenes started to grow after a lag phase of 3 and 10 days, respectively. When samples were stored at variable temperature conditions, L. monocytogenes numbers showed a lag phase of 5 days. CONCLUSIONS: Use of a conditioning liquid characterized by acidity and a correct storage temperature is able to counteract pathogen replication during shelf life. A high concentration of lactic acid bacteria was associated with effective control of L. monocytogenes but the role of lactic acid bacteria in WBMC conditioning liquid requires further investigation. SIGNIFICANCE AND IMPACT OF THE STUDY: According to European regulations, food producers should be able to justify decision-making on the shelf life assigned to their products, taking into account reasonable storage conditions and use by consumers. The results of the trial yielded information for producers of WBMC and similar cheeses for decision-making on product shelf life.

Isolation of glucosidase and phospholipase positive Bacillus circulans on ALOA medium.

AIM: To report the growth of glucosidase and phospholipase positive bacteria on agar Listeria according to Ottaviani and Agosti (ALOA) different from Listeria monocytogenes, Listeria ivanovii and Bacillus cereus. METHODS AND RESULTS: Raw water-buffalo milk was analysed according to EN ISO 11290. Streaking of Fraser broth on ALOA resulted in green colonies with an opaque halo after 48 h at 30°C. Colonies were transferred onto Tryptone soya yeast extract agar and showed cultural characteristics atypical for L. monocytogenes. Results of confirmation tests according to EN ISO 11290 method: negative haemolysis test, weak positive camp test in correspondence with Staphylococcus aureus, no fermentation of rhamnose, fermentation of xylose. Gram staining showed tapered, curved, Gram-positive rods with subterminal to terminal ellipsoidal spores, 0.5-0.7 µm diameter 4-5 µm. API 50CH CHB kit (99.9% percentage of identification) and the sequence of the 833 bp PCR product (portion of 16S rRNA, generic primers 1492-r; p27-f) showed 97% identity with Bacillus circulans ATCC 4513 (GenBank AY724690). CONCLUSIONS: Some B. circulans strains can grow on ALOA, according to ISO 11290, confirmation tests readily differentiate B. circulans from L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The different morphology of the colonies must be kept in mind to select true L. monocytogenes for confirmation test and to avoid overestimation of L. monocytogenes count.