Occurrence of a New Variant of Salmonella Infantis Lacking Somatic Antigen.

Salmonella Infantis is one of the most frequent serovars reported in broilers and is also regularly identified in human salmonellosis cases, representing a relevant public health problem. In the laboratories of the Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (IZSPB), six Salmonella Infantis strains with antigenic formula -:r:1,5 have been isolated from the litter and carcass of broilers between 2018 and 2022. The strains were investigated to evaluate their phenotype, antibiotic resistance and genomic profiles. Genomic analysis confirmed that the isolates belonged to the Infantis serotype and to the sequence type ST32. Moreover, all strains showed a multidrug-resistant (MDR) profile and were characterised by the presence of the IncFIB plasmid incompatibility group. Three strains had the blaCTX-M-1 gene, and one of them carried IncX1. The presence of this new variant of S. Infantis is particularly relevant because it could expand the landscape of the S. Infantis population. The absence of the somatic antigen could pose a problem in both isolation and serotyping and a consequent public health concern due to the spread of Salmonella infection.

Detection of Potential Zoonotic Agents Isolated in Italian Shelters and the Assessment of Animal Welfare Correlation with Antimicrobial Resistance in Escherichia coli Strains.

Welfare conditions in shelters, where dogs might be housed for a long period of time, may have a possible correlation with the occurrence of bacterial pathogens and their antimicrobial resistance (AMR). In this study, we assessed the occurrence of AMR in 54 strains of Escherichia coli isolated from dogs housed in 15 Italian shelters and we correlated the resistance patterns to animal welfare. We also aimed to evaluate the presence of specific pathogens with zoonotic potential in sheltered dogs. Thus, nasopharyngeal, rectal, and oral swabs were collected from a group of 20 dogs in each shelter and totaled 758 swabs. We identified 9 Staphylococcus pseudointermedius, 1 Pasteurella multocida, 9 Staphylococcus aureus, 12 Campylobacter spp., 54 Escherichia coli, 2 Salmonella enterica, and 246 Capnocytophaga spp. The antimicrobial susceptibility was assessed for the E. coli isolates using a panel of 14 antibiotics. The highest level of relative AMR was recorded for ampicillin and sulfamethoxazole. The association found between AMR and the levels of animal welfare scores in shelters was evident although not statistically significant. These results support the hypothesis that the good management of shelters can increase the level of animal welfare, thus reducing the use of antibiotics and, as a consequence, the AMR occurrence found in dogs that share their domestic environment with humans.

Systematic Survey of Vibrio spp. and Salmonella spp. in Bivalve Shellfish in Apulia Region (Italy): Prevalence and Antimicrobial Resistance.

The emergence of antimicrobial resistance (AMR) is increasingly common across the globe and aquatic ecosystems could be considered a reservoir of antibiotic-resistant bacteria. This study aimed to determine prevalence and antibiotic susceptibility of the potential pathogenic bacteria Salmonella spp. and Vibrio spp. in bivalve molluscs intended for human consumption, collected over a period of 19 months along the northern coast of Apulia region. The AMR profile was also determined in non-pathogenic Vibrio species, common natural inhabitants of seawater and a useful indicator for the surveillance of AMR in the environment. The current study presents data on the AMR of 5 Salmonella and 126 Vibrio isolates by broth microdilution MIC. Multidrug resistance (MDR) was observed in one S. Typhimurium strain towards sulfamethoxazole, trimethoprim, tetracycline, gentamicin, and ampicillin and in 41.3% of the Vibrio strains, mostly towards sulphonamides, penicillin, and cephems. All Vibrio isolates were sensitive to azithromycin, chloramphenicol, tetracycline, amoxicillin/clavulanic acid, gentamicin, streptomycin, amikacin, and levofloxacin. The AMR phenomenon in the investigated area is not highly worrying but not entirely negligible; therefore, in-depth continuous monitoring is suggested. Results concerning the antibiotic agents without available specific clinical breakpoints could be useful to upgrade the MIC distribution for Vibrio spp. but, also, the establishment of interpretative criteria for environmental species is necessary to obtain a more complete view of this issue.

Evaluation of Antimicrobial Resistance in Salmonella Strains Isolated from Food, Animal and Human Samples between 2017 and 2021 in Southern Italy.

Salmonella enterica is one of the most common causes of foodborne infection in the world, and the most common one in Italy. Italy collaborates with the other EU member states to survey the antimicrobial resistance of Salmonella on a large scale. This study on the situation in Apulia and Basilicata provides a more focused point of view on the territory, and anticipates the data reported in future Italian reports. Antimicrobial resistance was detected using the MIC detection method, with EUVSEC® plates, on the strains collected between 2017 and 2021. The results of serotyping showed that Salmonella Infantis is the serovar that has increased the most over time in veterinary samples, while Salmonella Tyhimurium and its monophasic variant are the most isolated in human samples. The results of the antimicrobial resistance study comply with European data, showing high resistance to quinolones, tetracyclines, ampicillin and trimethoprim, and low resistance to colistin and cephems. The significant exception was that all strains were resistant to sulphametoxazole. The presence of MDRs, which was 85% in veterinary and 77.4% in human strains, often included critically important antibiotics, which is a sign that more study and action is needed to manage the use of antibiotics.

Campylobacter jejuni and Campylobacter coli: prevalence, contamination levels, genetic diversity and antibiotic resistance in Italy.

A research was carried out in Italy with the aim of assessing Campylobacter contamination in broilers from breeding to slaughter, of defining the genetic diversity of isolates and their antibiotic resistance. Sampling was carried out in a slaughterhouse, and in farms representative of the most common broiler production in Italy. At farm, the 78.8% (95% C.I.: 74.5%­82.5%) of cloacal samples tested positive for Campylobacter spp. C. jejuni showed higher prevalence in winter than in spring and summer (p < 0.00001, χ2 = 32.9), while C. coli showed an opposite trend (p < 0.00001, χ2= 41.1). At slaughterhouse, the 32.3% (95% C.I.: 30.2%­35.2%) and the 23.9% (95% C.I.: 21.7%­26.3%) of skin samples tested positive for C. jejuni for C. coli, respectively. C. coli showed higher prevalence than C. jejuni at washing (p < 0.05, χ2 = 11.11) and at chilling (p < 0.05, χ2 = 9.26). PFGE revealed high heterogeneity among isolates. Some clones were identified within the same farm in more than one season, suggesting environmental conditions able to support their persistence; other clones resulted to be spatially distant, suggestive of cross­contamination. Both Campylobacter species showed high resistance to nalidixic acid and ciprofloxacin, while resistance to erythromycin was more frequent in C. coli than C. jejuni (p < 0.05; χ2 test).

Prevalence of netB-positive Clostridium perfringens in Italian poultry flocks by environmental sampling.

Clostridium perfringens type G is one of the pathogens involved in enteric diseases in poultry. NetB, a pore-forming toxin, is considered the main virulence factor responsible for necrotic enteritis during C. perfringens infection. We carried out a field study involving 14 farms to evaluate the occurrence of netB-positive C. perfringens and the impact of infection in Italian poultry flocks. Environmental samples (n = 117) and 50 carcasses were screened by microbiologic and molecular methods. Microbiologic investigations yielded 82 C. perfringens isolates. DNA was extracted from all samples and screened for α-toxin and NetB encoding genes by real-time PCR. The C. perfringens α-toxin gene was detected in 151 of 167 extracts (90.4%), and 31 of 151 (20.5%) were netB gene positive also. Sixteen isolates from a turkey flock with mild enteric disorders were also netB positive, demonstrating their occurrence not only in broiler but also in turkey flocks. A pulsed-field gel electrophoresis protocol was optimized to evaluate the diversity among isolates and revealed high genetic heterogeneity. The complete NetB toxin-coding gene of 2 C. perfringens isolates from turkey and broiler flocks were analyzed and showed very high relatedness with analogous sequences worldwide.

Correction: Antimicrobial resistance genotypes and phenotypes of Campylobacter jejuni isolated in Italy from humans, birds from wild and urban habitats, and poultry.

[This corrects the article DOI: 10.1371/journal.pone.0223804.].

Antimicrobial resistance genotypes and phenotypes of Campylobacter jejuni isolated in Italy from humans, birds from wild and urban habitats, and poultry.

Campylobacter jejuni, a common foodborne zoonotic pathogen, causes gastroenteritis worldwide and is increasingly resistant to antibiotics. We aimed to investigate the antimicrobial resistance (AMR) genotypes of C. jejuni isolated from humans, poultry and birds from wild and urban Italian habitats to identify correlations between phenotypic and genotypic AMR in the isolates. Altogether, 644 C. jejuni isolates from humans (51), poultry (526) and wild- and urban-habitat birds (67) were analysed. The resistance phenotypes of the isolates were determined using the microdilution method with EUCAST breakpoints, and AMR-associated genes and single nucleotide polymorphisms were obtained from a publicly available database. Antimicrobial susceptibility testing showed that C. jejuni isolates from poultry and humans were highly resistant to ciprofloxacin (85.55% and 76.47%, respectively), nalidixic acid (75.48% and 74.51%, respectively) and tetracycline (67.87% and 49.02%, respectively). Fewer isolates from the wild- and urban-habitat birds were resistant to tetracycline (19.40%), fluoroquinolones (13.43%), and quinolone and streptomycin (10.45%). We retrieved seven AMR genes (tet (O), cmeA, cmeB, cmeC, cmeR, blaOXA-61 and blaOXA-184) and gyrA-associated point mutations. Two major B-lactam genes called blaOXA-61 and blaOXA-184 were prevalent at 62.93% and 82.08% in the poultry and the other bird groups, respectively. Strong correlations between genotypic and phenotypic resistance were found for fluoroquinolones and tetracycline. Compared with the farmed chickens, the incidence of AMR in the C. jejuni isolates from the other bird groups was low, confirming that the food-production birds are much more exposed to antimicrobials. The improper and overuse of antibiotics in the human population and in animal husbandry has resulted in an increase in antibiotic-resistant infections, particularly fluoroquinolone resistant ones. Better understanding of the AMR mechanisms in C. jejuni is necessary to develop new strategies for improving AMR programs and provide the most appropriate therapies to human and veterinary populations.

Tracing Back Clinical Campylobacter jejuni in the Northwest of Italy and Assessing Their Potential Source.

Food-borne campylobacteriosis is caused mainly by the handling or consumption of undercooked chicken meat or by the ingestion of contaminated raw milk. Knowledge about the contributions of different food sources to gastrointestinal disease is fundamental to prioritize food safety interventions and to establish proper control strategies. Assessing the genetic diversity among Campylobacter species is essential to our understanding of their epidemiology and population structure. We molecularly characterized 56 Campylobacter jejuni isolates (31 from patients hospitalized with gastroenteritis, 17 from raw milk samples, and 8 from chicken samples) using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) in order to trace the source of the disease. We also used a population genetic approach to investigate the source of the human cases from six different reservoirs of infection. MLST identified 25 different sequence types and 11 clonal complexes (CCs) (21, 658, 206, 353, 443, 48, 61, 257, 1332, 354, 574) and these included several alleles not cited previously in the PubMLST international database. The most prevalent CCs were 21, 206, and 354. PFGE showed 34 pulsotypes divided between 28 different clusters. At the fine scale, by means of PFGE and MLST, only two human cases were linked to raw milk, while one case was linked to chicken meat. The investigation revealed the presence of several genotypes among the human isolates, which probably suggests multiple foci for the infections. Finally, the source attribution model we used revealed that most cases were attributed to chicken (69.75%) as the main reservoir in Italy, followed to a lesser extent by the following sources: cattle (8.25%); environment (6.28%); wild bird (7.37%); small ruminant (5.35%), and pork (2.98%). This study confirms the importance of correlating epidemiological investigations with molecular epidemiological data to better understand the dynamics of infection.

Population Diversity of Campylobacter jejuni in Poultry and Its Dynamic of Contamination in Chicken Meat.

This study aimed to analyse the diversity of the Campylobacter jejuni population in broilers and to evaluate the major source of contamination in poultry meat. Eight rearing cycles over one year provided samples from three different broiler farms processed at the same slaughterhouse. A total of 707 C. jejuni were isolated from cloacal swabs before slaughter and from the breast skin of carcasses after slaughter and after chilling. All suspected Campylobacter colonies were identified with PCR assays and C. jejuni was genotyped by sequence analysis of the flaA short variable region (SVR) and by pulsed-field gel electrophoresis (PFGE) using SmaI enzyme. Phenotypic antibiotic resistance profiles were also assayed using minimal inhibitory concentration (MIC). The flocks carried many major C. jejuni clones possibly carrying over the rearing cycles, but cross contamination between farms may happen. Many isolates were resistant to fluoroquinolones, raising an issue of high public concern. Specific Campylobacter populations could be harboured within each poultry farm, with the ability to contaminate chickens during each new cycle. Thus, although biosecurity measures are applied, with a persistent source of contamination, they cannot be efficient. The role of the environment needs further investigation to better address strategies to control Campylobacter.

Molecular typing of Salmonella enterica subspecies enterica serovar Typhimurium isolated in Abruzzo region (Italy) from 2008 to 2010.

In this study, 47 antibiotic-resistant strains of Salmonella enterica subspecies enterica serovar Typhimurium (ST) were characterised, including 15 monophasic variants 1, 4, [5], 12:i:-, (STm) isolated from different matrices. They were all selected from 389 Salmonella enterica subspecies enterica strains isolated during 2008-2010 in Abruzzo region (Italy). Thirty-seven strains showed to be resistant to more than 1 antibiotic. Among 47 isolates, phage type U311 and DT104 were identified. The ASSuT resistance pattern was predominant in mST strains and ACSSuT in ST DT104 and U302. A multiplex Polimerase Chain Reaction (PCR) method was used to investigate 4 genes: fluorfenicol (floSt), virulence (spvC), invasine (invA) and integrase (int). All ST the strain were positive for invA gene and 28,32% of strains were positive for spvC gene. PFGE analysis revealed a large number of small clonal populations, however not ascrivable to outbreaks.

Characterization of antimicrobial resistance patterns and detection of virulence genes in Campylobacter isolates in Italy.

Campylobacter has developed resistance to several antimicrobial agents over the years, including macrolides, quinolones and fluoroquinolones, becoming a significant public health hazard. A total of 145 strains derived from raw milk, chicken faeces, chicken carcasses, cattle faeces and human faeces collected from various Italian regions, were screened for antimicrobial susceptibility, molecular characterization (SmaI pulsed-field gel electrophoresis) and detection of virulence genes (sequencing and DNA microarray analysis). The prevalence of C. jejuni and C. coli was 62.75% and 37.24% respectively. Antimicrobial susceptibility revealed a high level of resistance for ciprofloxacin (62.76%), tetracycline (55.86%) and nalidixic acid (55.17%). Genotyping of Campylobacter isolates using PFGE revealed a total of 86 unique SmaI patterns. Virulence gene profiles were determined using a new microbial diagnostic microarray composed of 70-mer oligonucleotide probes targeting genes implicated in Campylobacter pathogenicity. Correspondence between PFGE and microarray clusters was observed. Comparisons of PFGE and virulence profiles reflected the high genetic diversity of the strains examined, leading us to speculate different degrees of pathogenicity inside Campylobacter populations.

Epidemiological study of an outbreak of Norovirus in a rest home in Italy.

A Norovirus GII.4 (variant 2006) epidemic occurred in a rest home in the Abruzzo region of Italy between January and March 2009. Rest homes are typologically exposed to Norovirus infections as often reported in the literature. The investigation proposed in this article focused on identifying the source of infection and the route of propagation of the virus in the infected rest home. Microbiological, chemical and hygiene/health investigations were conducted on residents and employees of the home and on its water supply. A questionnaire was designed and distributed to identify the habits of the subjects who promoted the spread of the virus, in order to obtain a retrospective view of the event.

Detection and genotyping of Campylobacter jejuni and Campylobacter coli by use of DNA oligonucleotide arrays.

Campylobacter have emerged as the most common bacterial food-borne illness in the developed world. The ability to reduce Campylobacter infections in humans is linked to the full comprehension of the principal key aspects of its infection cycle. A microbial diagnostic microarray detecting Campylobacter housekeeping, structural, and virulence associated genes was designed and validated using genomic DNA from reference and field strains of Campylobacter jejuni and coli isolated from human, chicken, and raw milk. This microarray was confirmed to be a powerful diagnostic tool for monitoring emerging Campylobacter pathotypes as well as for epidemiological, environmental, and phylogenetic studies including the evaluation of genome plasticity.

Prevalence of thermotolerant Campylobacter in broiler flocks and broiler carcasses in Italy.

In accordance with European Union regulations, from 5 February until 15 December 2008, sampling and analysis activities were conducted in Italy to assess the extent of contamination caused by thermotolerant Campylobacter in broiler chickens farmed nationwide. The survey involved 48 poultry slaughterhouses distributed across eleven regions of Italy, where the caeca and carcasses of 393 slaughter batches were sampled. A total of 284 batches (72.3%) gave positive results for Campylobacter spp. as follows: 52.1% were contaminated by C. jejuni, 55.6% by C. coli and 1.1% by C. lari. C. jejuni and C. coli were isolated together in 37 batches (13% of positive results). Campylobacter spp. was isolated only from the caeca in 251 slaughter batches (63.9%) including caecal isolates of C. jejuni (48.2%), C. coli (50.6%), and C. lari (1.2%). Carcasses from 182 batches (46.3%) were contaminated by C. jejuni in 40.7% of cases, C. coli in 57.7% and the absence of C. lari from all batches examined. The contamination level observed in the carcasses ranged between 10 and 1.6 × 10(7) cfu/g.

Polymerase chain reaction and bacteriological comparative analysis of raw milk samples and buffalo mozzarella produced and marketed in Caserta in the Campania region of Italy.

To help identify an epidemiological link between the consumption of buffalo mozzarella prepared with raw milk (not heat-treated) and cases of human brucellosis, 80 samples of raw buffalo milk and 315 samples of mozzarella were tested for the presence of Brucella spp. Samples originated from Caserta, the province with the highest number of Brucella-positive buffalo herds in Campania, the region in which 96.02% of all cases of human brucellosis reported in 2000-2005 were recorded. To take into account possible seasonal variations, between February 2006 and March 2007, samples were purchased directly from 72 dairy outlets that were representative of the province. Samples were tested for Brucella spp. using the polymerase chain reaction (PCR) and bacterial isolation. Samples tested negative for Brucella using both methods. Spiked samples were then prepared and tested by PCR and bacterial isolation and the sensitivity, specificity, repeatability, reproducibility and limit of detection were determined. The PCR limit of detection was below 1 colony-forming unit (cfu)/g. The repeatability and reproducibility of the method were 100% (p = 0.95), the sensitivity was 96.7% (p = 0.95) and the specificity was 100% (p = 0.95).

Preliminary investigations into fluoroquinolone resistance in Escherichia coli strains resistant to nalidixic acid isolated from animal faeces.

Resistance to fluoroquinolones was evaluated in 17 nalidixic acid-resistant Escherichia coli strains isolated from animal faeces. Polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), hybridisation and sequencing were used to reveal point mutations in DNA gyrase subunits A and B and the presence of the qnr gene as evidence of plasmid resistance. Chromosomal resistance and class 1 integrons were found in 10 of the 17 E. coli strains examined, while the qnr gene was not found in any of these. Mutation Ser83-Leu was found in six strains and in one strain mutation Ser83-Ala was found in gyrA. Mutations in gyrA Asp87 codons and in gyrB Asp426 or Lys 447 codons were not identified. Sequencing of the E. coli strain ATCC 25922 subjected to resistance induction revealed a gyrB mutation of the Lys 447 residue which had been replaced by arginine.

Typing of Brucella field strains isolated from livestock populations in Italy between 2001 and 2006.

The identification of species and biovars of Brucella field strains isolated in outbreaks is essential to fully understand the epidemiology of the disease and to trace sources of infection, thereby improving the outcome of brucellosis eradication programmes. It is important to identify the presence of Brucella strains in livestock populations and to determine the presence of new strains that might previously have been considered exotic. In this study, 732 Brucella strains isolated from livestock were submitted for typing by the Italian Istituti Zooprofilattici Sperimentali to the National Reference Laboratory for brucellosis between 2001 and 2006. Animal species affected, biovars typed and spatial distribution of isolates are discussed. Brucella field strains were identified using both conventional bacteriological methods and molecular techniques. Species identification was performed using the AMOS (AbortusMelitensis OvisSuis)-polymerase chain reaction. For biovar identification, amplification of omp2a, omp2b and omp31 genes was performed, followed by restriction fragment length polymorphism. Final biovar identification was performed by growth in the presence of basic fuchsin and thionin, using the slide agglutination test with Brucella A- and M- specific antisera.