Antimicrobial Resistance in Physiological and Potentially Pathogenic Bacteria Isolated in Southern Italian Bats.

The spread of antimicrobial resistance is one of the major health emergencies of recent decades. Antimicrobial-resistant bacteria threaten not only humans but also populations of domestic and wild animals. The purpose of this study was to evaluate the distribution of antibiotic resistance (AMR) and multidrug resistance (MDR) in bacterial strains isolated from six Southern-Italian bat populations. Using the disk diffusion method, we evaluated the antimicrobial susceptibility of 413 strains of Gram-negative bacteria and 183 strains of Gram-positive bacteria isolated from rectal (R), oral (O) and conjunctival (C) swabs of 189 bats belonging to 4 insectivorous species (Myotis capaccinii, Myotis myotis, Miniopterus schreibersii and Rhinolophus hipposideros). In all bat species and locations, numerous bacterial strains showed high AMR levels for some of the molecules tested. In both Gram-negative and Gram-positive strains, the resistance patterns ranged from one to thirteen. MDR patterns varied significantly across sites, with Grotta dei Pipistrelli in Pantalica displaying the highest levels of MDR (77.2% of isolates). No significant differences were found across different bat species. Monitoring antibiotic resistance in wildlife is a useful method of evaluating the impact of anthropic pressure and environmental pollution. Our analysis reveals that anthropic contamination may have contributed to the spread of the antibiotic resistance phenomenon among the subjects we examined.

Cultivable Bacteria Associated with the Microbiota of Troglophile Bats.

BACKGROUND: The study of bats is of significant interest from a systematic, zoogeographic, ecological, and physiological point of view. The aim of this study is to investigate the culturable aerobic enteric, conjunctival, and oral bacterial flora of bats to determine their physiological microbiome and to investigate the possible occurrence of pathogenic bacteria. METHODS: Five hundred and sixty-seven samples were collected from 189 individuals of four species of troglophile bats (Myotis myotis, Myotis capaccinii, Miniopterus schreibersii, and Rhinolophus hipposideros) living in Sicilian and Calabrian territory (Italy). All samples were tested for Gram-negative bacteria; conjunctival and oral swabs were also submitted to bacteriological examination for Gram-positive bacteria. RESULTS: Four hundred thirteen Gram-negative strains were isolated. Of these, 377 belonged to 17 different genera of the family Enterobacteriaceae and 30 to five other families. One hundred eighty-three Gram-positive strains were isolated. Of these, 73 belonged to the Staphylococcaceae family, 72 to the Bacillaceae family and 36 to four other families. Besides some potentially pathogenic strains, several bacterial species have been found that are common to all the bat species studied. These could perhaps play a physiological or nutritional role. CONCLUSION: A great variety of bacterial species were identified in the cultivable microbiota of southern-Italian troglophile bats, including several potentially pathogenic strains and numerous putatively symbiotic species.

Phenotypic and genotypic study on antibiotic resistance and pathogenic factors of Staphylococcus aureus isolates from small ruminant mastitis milk in South of Italy (Sicily).

Staphyloccoccus aureus is the major cause of mastitis in small ruminants in the Mediterranean farms causing severe losses to dairy industry. Antibiotic treatment has been the most common approach to control these infections. Aim of this study was to investigate antimicrobial resistance (AMR), virulence factors and biofilm-related genes of 84 Sicilian strains of S. aureus isolated from sheep and goats milk during two different periods δT1 (2006-2009) and δT2 (2013-2015). Kirby Bauer method and Polymerase Chain Reaction (PCR) were utilized to monitor AMR and related genes (mecA, tetK, tetM, ermA, ermC). Moreover, toxin genes (tsst-1, sea-see, seg-sej, and sep) and biofilm genes (bap, ica, sasC) were studied. Twenty-six isolates (30.9%) showed multidrug resistance. The two groups showed similar results with exception for higher values of resistance for tilmicosin and lower for sulfamethoxazole and vancomycin of the second group. MecA gene was detected in one isolate. Tetracycline resistance was higher than 20%, with an increase in δT2 group. Toxin genes were found in 5 isolates (5.9%), belonging of δT2 group, while 57 of isolates (67.8%) showed biofilm related genes. The high presence of multi-resistant isolates suggests the need of more responsible use of antibiotic therapy for the control of these infections.

Analysis of Eurasian Stone curlew (Burhinus oedicnemus) microbial flora reveals the presence of multi-drug resistant pathogens in agro-pastoral areas of Sicily (Italy).

Spread of multi-drug resistant (MDR) bacteria in natural environments pose a risk to human and animal health. Wild birds are considered to be reservoirs of human pathogens and vectors of antimicrobial resistance distribution in the environment. The aim of this study is to assess the occurrence of antibiotic resistant bacteria in isolates from bird specimens living in three agro-pastoral areas of the southeastern Sicily. We analyzed the microbiomes of the Eurasian Stone curlew Burhinus oedicnemus (Charadriiformes, Aves) and identified 91 Gram positive and 212 Gram negative strains, whose antimicrobial susceptibility to 11 and 9 antibiotic classes (respectively) was evaluated using agar disk diffusion test. Isolates showed significant levels of antimicrobial resistance, and a high percentage of MDR strains was found both between the Gram positive (49.4%) and the Gram negative (34.9%). Multi-drug resistance levels are higher among strains isolated in the beak and the eye than among enteric (faeces and cloaca) strains. Our results indicate high levels of MDR strains among wild bird populations, with a potential threat to wildlife and human populations.

Detection of multidrug resistance and extended-spectrum/plasmid-mediated AmpC beta-lactamase genes in Enterobacteriaceae isolates from diseased cats in Italy.

OBJECTIVES: The aim of this study was to determine the antimicrobial susceptibility of Enterobacteriaceae isolated from cats affected by diseases commonly encountered in practice, and to characterise the third-generation cephalosporin (3GC)-resistance molecular mechanisms involved. METHODS: Clinical samples (n = 100) included 58 rectal swabs from cats with diarrhoea, 31 nasal swabs from cats with clinical signs of upper respiratory tract disease, four ear swabs from cats with otitis, three conjunctival swabs from cats with conjunctivitis, two oral swabs from cats with stomatitis, one swab from a skin abscess and one urine sample from a cat with cystitis. A total of 125 Enterobacteriaceae were isolated from 90 cats. Escherichia coli was the most frequently isolated species (n = 65), followed by Enterobacter species (n = 20), Proteus species (n = 13), Citrobacter species (n = 12) and others (n = 15). Bacterial susceptibility testing was performed with respect to eight antimicrobial classes. Beta (ß)-lactamase genes were identified by PCR and nucleotide sequencing. RESULTS: Overall, the higher frequency of resistance was to amoxicillin-clavulanate (61.3%), trimethoprim/sulfamethoxazole (33.6%) and cefotaxime (32.8%). Thirty-six percent of the isolates (n = 45) were resistant to 3GCs. Of these isolates, 34 were tested by PCR and nucleotide sequencing and 23 were confirmed as encoding ß-lactamase genes. Fourteen 3GC-resistant isolates harboured extended-spectrum ß-lactamases (ESBLs) belonging to groups CTX-M-1 (n = 12, two of which were CTX-M-79), CTX-M-2 (n = 1) and CTX-M-9 (n = 1), as well as SHV-12 (n = 1) and TEM-92 (n = 1). Nine isolates had CMY-2 plasmid-mediated AmpC ß-lactamases (pAmpC). Thirty-one percent (n = 39) of the isolates were multidrug resistant (MDR) and were isolated from 34% (n = 31/90) of the cats. CONCLUSIONS AND RELEVANCE: A high frequency of MDR and ESBL/pAmpC ß-lactamase-producing Enterobacteriaceae were detected among bacteria isolated from a feline population in southern Italy with a variety of common clinical conditions, which poses limitations on therapeutic options for companion animals. We describe the first detection of CTX-M-79 and TEM-92 ESBL genes in isolates from cats.

Study of the spread of antimicrobial-resistant Enterobacteriaceae from wild mammals in the National Park of Aspromonte (Calabria, Italy).

Antimicrobial-resistant (AMR) bacteria are a threat to global health. Epidemiological studies are necessary to gain a more detailed understanding of the transmission modality of resistant bacteria to wild animals and their diffusion into the environment to ensure a broader evaluation of the epidemiological role of wildlife. The study aimed to identify little-known epidemiological aspects by focusing on the isolation of Enterobacteriaceae in order to bring knowledge on the bacterial flora of wild mammals living in the National Park of Aspromonte (Calabria, Italy). Two hundred twenty-five faecal samples of wild mammals were collected and submitted to standard bacteriological examination for Enterobacteriaceae detection. Isolates were identified by mass spectrometry MALDI-TOF (matrix assisted laser desorption/ionisation - time of fligt mass spectrometry). The 30 isolates showing the highest number of resistances were screened for Extended Spectrum Beta Lactamase (ESBL) production. Antimicrobial susceptibility testing of the bacterial isolates was performed by the disk diffusion method. Two hundred sixty-three strains were isolated. The most frequently recovered bacterial species were Hafnia alvei, Escherichia coli and Citrobacter spp.. Potentially pathogenic species including Salmonella spp., Yersinia enterocolitica and Serratia marcescens have also been identified. Isolates displayed significant frequencies of antibiotic resistance. The resistance to amoxicillin (71.4%) was the most frequent, followed by amoxicillin/clavulanic acid (55.5%), ampicillin (49.8%), streptomycin (35.7%), doxycycline (16.3%), tobramycin (15.2%), tetracycline (13.3%) and gentamicin (10.6%). Nine strains showed resistance to imipenem and 1 to meropenem. All isolates were negative for the presence of ESBL-encoding genes. Wild animals can be regarded as sentinel species and used as environmental health indicators.

Antimicrobial resistance patterns of Enterobacteriaceae in European wild bird species admitted in a wildlife rescue centre.

Wild birds have been considered to be reservoirs of enteric human pathogens and vectors of resistance dissemination to the environment. During annual migration, they potentially play a role in the epidemiology of human associated zoonoses. The aim of this study was to investigate the frequency of isolation and antimicrobial susceptibility profiles of Enterobacteriaceae members isolated from cloacal swabs of common European wild birds. Fifty-five cloacal swabs were taken during birds' entrance evaluation in a rescue centre for injured wild birds in the Province of Messina (Sicily, Italy). All samples were examined for the presence of members of the family Enterobacteriaceae using standard methods and on the isolated strains antibiotic susceptibility testing was performed. Eighty three Enterobacteriaceae strains were isolated from raptors, waterbirds and passerines. The bacterial species isolated were: Escherichia coli, Proteus mirabilis, Proteus vulgaris, Citrobacter freundii, Enterobacter cloacae, Klebsiella oxytoca, Salmonella Typhimurium, Escherichia vulneris, Enterobacter amnigenus biogroup 2, Salmonella Duesseldorf and Hafnia alvei. The isolates showed significant frequencies of antibiotic resistance. Multiresistance to three or more groups of antibiotics also occurred. None of them have shown a phenotypic Extended Spectrum Beta Lactamase (ESBL) profile.

Detection of methicillin-resistant Staphylococcus aureus (MRSA) in the microbial flora from the conjunctiva of healthy donkeys from Sicily (Italy).

OBJECTIVE: To describe the bacterial flora present in the normal conjunctiva of donkeys from Sicily (Italy). ANIMALS STUDIED: A total of 46 healthy donkeys housed in 3 locations within the territory of Palermo (Sicily, Italy) were studied. Donkeys ranged from 2 to 13 years of age, with a median age of 6 years. PROCEDURES: Forty-six conjunctival swabs were obtained from both eyes of each animal, and specimens were cultured for aerobic bacteria. Furthermore, the antimicrobial activity of methicillin (1 µg) and oxacillin (5 µg) on Staphylococcus spp. isolates was evaluated, and a specific PCR assay, which allows the detection of mecA gene specific for methicillin-resistant Staphylococcus aureus (MRSA) strains, was performed. RESULTS: Forty of 46 (86.9%) donkeys were positive for bacteria. Eighty bacterial isolates, representing 9 bacteria genera, were successfully cultured. The most frequently recovered bacterial genus was Staphylococcus (52/80 isolates; 65%). Several strains (20/80 isolates; 25%) belonging to the Enterobacteriaceae family were also isolated, among which the most frequently isolated genus was Enterobacter (eight isolates). Of the 52 Staphylococcus spp. isolates, 14 (26.9%) strains were oxacillin/methicillin resistant. The mecA gene was detected in 6/52 (11.5%) strains. CONCLUSIONS: This study contributes to the knowledge about normal ocular flora and MRSA occurrence in donkey farms in Sicily.

Pathogenic microorganisms carried by migratory birds passing through the territory of the island of Ustica, Sicily (Italy).

Several studies have shown that migratory birds play an important role in the ecology, circulation and dissemination of pathogenic organisms. In October 2006, a health status evaluation was performed on a large population of migratory birds passing through the territory of Ustica (Italy), an island located on the migration route of many species of birds to Africa, and various laboratory tests were conducted. In total, 218 faecal swabs and the internal organs of 21 subjects found dead in nets were collected for bacteriological and virological examination, including avian influenza and Newcastle disease. In addition, 19 pooled fresh faecal samples were collected for mycological examination. The bacteriological analysis produced 183 strains belonging to 28 different species of the Enterobacteriaceae family. In particular, Salmonella bongori, Yersinia enterocolitica and Klebsiella pneumonia strains were isolated. Almost all of the isolates were susceptible to sulphamethoxazole/trimethoprime (99.4%), cefotaxime (98.9%), nalidixic acid (96.7%), chloramphenicol (95.6%), and tetracycline (93.4%). Alternatively, many strains were resistant to ampicillin (42.6%), amoxicillin-clavulanic acid (42.6%), and streptomycin (43.7%). According to reverse transcriptase-polymerase chain reaction analysis, all of the samples were negative for the M gene of avian influenza virus. Moreover, isolation tests conducted on specific pathogen free eggs were negative for avian influenza and Newcastle disease. Several hyphomycetes and yeasts belonging to different genera were present in the specimens, and Cryptococcus neoformans was observed in a pooled faecal sample. Antibiotic resistance in wildlife can be monitored to evaluate the impact of anthropic pressure. Furthermore, migratory birds are potential reservoirs of pathogenic agents; thus, they can be regarded as sentinel species and used as environmental health indicators.