Whole genome sequence analysis of Brucella abortus isolates from various regions of South Africa

The availability of whole genome sequences in public databases permits genome-wide comparative studies of various bacterial species. Whole genome sequence-single nucleotide polymorphisms (WGS-SNP) analysis has been used in recent studies and allows the discrimination of various Brucella species and strains. In the present study, 13 Brucella spp. strains from cattle of various locations in provinces of South Africa were typed and discriminated. WGS-SNP analysis indicated a maximum pairwise distance ranging from 4 to 77 single nucleotide polymorphisms (SNPs) between the South African Brucella abortus virulent field strains. Moreover, it was shown that the South African B. abortus strains grouped closely to B. abortus strains from Mozambique and Zimbabwe, as well as other Eurasian countries, such as Portugal and India. WGS-SNP analysis of South African B. abortus strains demonstrated that the same genotype circulated in one farm (Farm 1), whereas another farm (Farm 2) in the same province had two different genotypes. This indicated that brucellosis in South Africa spreads within the herd on some farms, whereas the introduction of infected animals is the mode of transmission on other farms. Three B. abortus vaccine S19 strains isolated from tissue and aborted material were identical, even though they originated from different herds and regions of South Africa. This might be due to the incorrect vaccination of animals older than the recommended age of 4–8 months or might be a problem associated with vaccine production.SUPPLEMENTARY TABLES: Table S1: Sample order used in Bruce-ladder (A) and AMOS (B) multiplex PCR assays and the descriptive information of the gel images, Table S2: Clean unique variants of the South African strains (refer to Table 2 for the sample names (in column 1) and sample ID (in column 2)).The Gauteng Department of Agriculture and Rural Development (GDARD), National Research Foundation, South Africa and Institute of Tropical Medicine, Belgium.https://www.mdpi.com/journal/microorganismsam2022Veterinary Tropical Disease

Indagine preliminare sulla resistenza ai fluorochinoloni in ceppi di Escherichia coli resistenti all’acido nalidixico isolati da feci animali

E’ stata valutata la possibile resistenza ai fluorochinoloni in 17 ceppi di Escherichia coli resistenti all'acido nalidixico, isolati da feci animali. Sono state impiegate tecniche di PCR, RFLP, ibridazione, e sequenziamento allo scopo di evidenziare la presenza di mutazioni puntiformi nelle subunità A e B della DNA girasi e possibile la presenza del gene qnr a testimonianza di eventuale resistenza plasmidica. In 10 dei 17 ceppi di E. coli esaminati è stata evidenziata una resistenza di tipo cromosomico con la presenza di integroni di classe I e in nessuno di essi è stata evidenziata la presenza del gene qnr. In 6 ceppi è stata osservata la mutazione Ser83-Leu e in un ceppo la mutazione Ser83-Ala. Non sono state evidenziate le mutazioni note ai codoni Asp87 di gyrA e Asp426 e Asp477 di gyrB. Dal sequenziamento del ceppo di E. coli ATCC 25922 sottoposto ad induzione di resistenza è stata rilevata una mutazione in gyrB al residuo Lys 447 che è stato sostituito da una Arginina

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

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)

Whole Genome Sequencing for Tracing Geographical Origin of Imported Cases of Human Brucellosis in Sweden

Human infections with Brucella melitensis are occasionally reported in Sweden, despite the fact that the national flocks of sheep and goats are officially free from brucellosis. The aim of our study was to analyze 103 isolates of B. melitensis collected from patients in Sweden between 1994 and 2016 and determine their putative geographic origin using whole genome sequencing (WGS)-based tools. The majority of the strains were assigned to East Mediterranean and African lineages. Both in silico Multiple Loci VNTR (Variable Number of Tandem Repeats) Analysis (MLVA) and core genome Multilocus Sequence Typing (cgMLST) analyses identified countries of the Middle East as the most probable source of origin of the majority of the strains. Isolates collected from patients with travel history to Iraq or Syria were often associated with genotypes from Turkey, as the cgMLST profiles from these countries clustered together. Sixty strains were located within a distance of 20 core genes to related genotypes from the publicly available database, and for eighteen isolates, the closest genotype was different by more than 50 loci. Our study showed that WGS based tools are effective in tracing back the geographic origin of infection of patients with unknown travel status, provided that public sequences from the location of the source are available

Comparazione fra polymerase chain reaction e isolamento batteriologico in campioni di latte crudo e mozzarella di bufala prodotta in provincia di Caserta, regione Campania (Italia)

Per contribuire all’individuazione di un possibile nesso epidemiologico tra consumo di mozzarella di bufala preparata con latte crudo (non trattato al calore) e casi di brucellosi umana, sono stati analizzati, per ricerca di Brucella spp., 80 campioni di latte bufalino crudo e 315 campioni di mozzarella. Gli alimenti esaminati sono stati prelevati in caseifici della provincia di Caserta dove è presente la più alta concentrazione di allevamenti bufalini sierologicamente positivi alla brucellosi in Campania, regione che, nel periodo 2000-2005, ha registrato il 96,02% dei casi di brucellosi umana notificati in Italia. Al fine de valutare possibili variazioni stagionali, i campioni sono stati acquistati in 72 rivendite associate a caseifici nel periodo febbraio 2006-marzo 2007. La ricerca di Brucella spp. è stata effettuata utilizzando polymerase chain reaction (PCR) ed eseguendo contemporaneamente l’isolamento microbiologico. I campioni esaminati sono risultati negativi alla ricerca di Brucella con entrambi i metodi utilizzati. Sono stati, inoltre, definiti i parametri di sensibilità, specificità, ripetibilità, riproducibilità e il limite di rilevazione del metodo molecolare, esaminando campioni artificialmente contaminati, sia con metodo PCR sia con isolamento microbiologico classico. Il limite di rilevazione è risultato inferiore a 1 UFC/g, ripetibiltà e riproducibilità sono stati pari a 100% (p=0,95), sensibilità a 96,7% (p=0,95) e specificità a 100% (p=0,95)

Isolamento di Brucella suis biovariante 2 da un cinghiale in Abruzzo, Italia

Un cinghiale selvatico femmina, di circa due anni di età, è stato trovato morto dai Servizi Veterinari a Pianola di Roio a L'Aquila, Provincia situata nella Regione Abruzzo nell'Italia centrale. La carcassa è stata conferita all'Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G. Caporale" di Teramo per l'esecuzione dell'esame autoptico. Dai linfonodi sotto-mandibolari di questo esemplare è stato isolato un ceppo di Brucella suis biovariante 2. Questa è la prima segnalazione d'isolamento di B. suis nella Regione Abruzzo. Diversi autori hanno accettato, in passato, l'ipotesi che B. suis biovariante 2 sia stata introdotta in Italia attraverso l'importazione di lepri provenienti dai paesi europei in cui l'infezione è endemica nelle popolazioni selvatiche. Questa considerazione ha portato le autorità italiane a rafforzare i controlli esistenti sulle lepri selvatiche importate a scopo di ripopolamento. Tuttavia, attualmente, non è in vigore alcuna disposizione (né lo è stata in passato) per il controllo della brucellosi nei cinghiali movimentati, sia a livello nazionale che europeo. L'isolamento di B. suis biovariante 2 da cinghiali in altre Regioni italiane geograficamente distanti potrebbe suggerire che questa specie, piuttosto che le lepri importate, possa essere stata la fonte d'introduzione dell'infezione in tali aree. Le norme nazionali ed europee di gestione della brucellosi nella fauna selvatica dovrebbero essere indirizzate al controllo dello stato di salute dei cinghiali negli allevamenti prima delle movimentazioni o del rilascio, con l'obiettivo di prevenire la diffusione di questo patogeno in territori indenni

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

Genomic Comparison of Salmonella Enteritidis Strains Isolated from Laying Hens and Humans in the Abruzzi Region during 2018

Salmonellosis is a major cause of bacterial foodborne infection. Since 2016, an increased number of cases of gastroenteritis caused by Salmonella enterica serovar Enteritidis linked to eggs produced in Poland has been reported in Europe. In Italy, S. Enteritidis is one of the three most commonly reported serotypes, associated mainly with the consumption of contaminated eggs and derived products. In our work, we analysed 61 strains of S. Enteritidis obtained from humans and farms in the Abruzzi region, Italy, in 2018. We used Multiple-Loci Variable-Number Tandem Repeat (VNTR) analysis (MLVA)-based typing and Whole-Genome Sequencing (WGS) tools to identify closely related strains and perform cluster analysis. We found two clusters of genetically similar strains. The first one was present in the local farms and isolated from human cases and had single-linkage distance of no more than two core genes and less than five Single-Nucleotide Polymorphisms (SNPs). The second cluster contained strains isolated from humans and from a dessert (tiramisù) sample that shared identical core genome and were assigned the same SNP address. Cluster 2 isolates were found to be genetically similar to an S. Enteritidis strain from a multi-country outbreak linked to Polish eggs