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Objective Meat and eggs from chickens infected with Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Infantis are considered to be an important source of Salmonella infections for humans. In order to control Salmonella infections in chickens, basic biosecurity measures are taken in combination with inactivated or attenuated live vaccines. Apart from an adaptive immune response, some live vaccines also induce innate immune mechanisms that prevent or inhibit systemic invasion with homologous Salmonella serovars. It is unknown whether these invasion inhibition effects are also directed against heterologous Salmonella serovars. Furthermore, it is unclear whether the adaptive immune response after vaccination with a Salmonella Enteritidis phage type 4 live vaccine is also directed against other phage types of Salmonella Enteritidis and Typhimurium. Material and methods Specific pathogen-free day-old chicks were vaccinated orally with a commercially available Salmonella Enteritidis live vaccine. To test the invasion inhibition effect, the animals were challenged orally with a labelled Salmonella Typhimurium or Salmonella Infantis strain 1 day after vaccination. To demonstrate the adaptive immune response against non-phage type 4 Salmonella Enteritidis strains and a monophasic Salmonella Typhimurium strain, the chickens were challenged with Salmonella Enteritidis strains of phage types 1, 8 and 21 and a monophasic Salmonella Typhimurium strain (Definitive Type 193). After challenge, the abundance of the challenge strain in liver and cecal tissue was enumerated and compared with a corresponding control group. Results Findings showed that the live Salmonella Enteritidis vaccine inhibits systemic invasion after early infection with Salmonella Typhimurium and Salmonella Infantis. Furthermore, adaptive immunity against the tested non-phage type 4 Salmonella Enteritidis strains and the monophasic Salmonella Typhimurium strain was demonstrated. Conclusion and clinical relevance The results of this study demonstrate that vaccination with the Salmonella Enteritidis phage type 4 live vaccine significantly inhibits the invasion of Salmonella Typhimurium and Infantis. Furthermore, an adaptive immune response was also detected against non-phage type 4 Salmonella Enteritidis strains and a monophasic Salmonella Typhimurium strain.

Introduction: Cholera is an acute diarrhoeal disease caused by Vibrio cholerae (V.cholerae). Based on antigenic differences of O antigen, O1 serogroup can be divided into three serotypes. In addition, by performing various biochemical reactions, O1 Serogroup can be differentiated into two biotypes. Outbreaks of Cholera occur seasonally. It is associated with monsoon season, warm temperature, heavy rainfall and increased plankton population. Aim: The aim was to determine the trends in resistance pattern and phenotypic Pattern of Vibrio cholerae. Materials and Methods: A retrospective study was conducted during the period from June 2019-December 2019. Culture of Stool specimens were done on different agar media. Biotyping was done by conventional methods. Serotyping and phage typing was also done along with the Antibiotic susceptibility testing. Descriptive analysis was used and presented in terms of percentage. Results:V.cholerae was isolated in 72 patients and they belonged to serogroup O1 and biotype El Tor. The most common serotype was Ogawa. The predominant phage type were T2 by old scheme and T27 by new scheme of phage typing. The maximum number of V. cholerae isolates was seen in the month of November, 2019 followed by October, 2019. Conclusion: The phenotypic pattern and fluctuating seasonal trend of V. cholerae and antimicrobial resistance encourage the continued epidemiological and microbiological surveillance of the disease.

SUMMARYSalmonella Enteritidis has emerged as the most prevalent cause of human salmonellosis in Canada. Recent trends of S. Enteritidis subtypes and their potential sources were described by integrating Salmonella data from several Canadian surveillance and monitoring programmes. A threefold increase in S. Enteritidis cases from 2003 to 2009 was identified to be primarily associated with phage types 13, 8 and 13a. Other common phage types (4, 1, 6a) showed winter seasonality and were more likely to be associated with cases linked to international travel. Conversely, phage types 13, 8 and 13a had summer seasonal peaks and were associated with cases of domestically acquired infections. During agri-food surveillance, S. Enteritidis was detected in various commodities, most frequently in chicken (with PT13, PT8 and PT13a predominating). Antimicrobial resistance was low in human and non-human isolates. Continued integrated surveillance and collaborative prevention and control efforts are required to mitigate future illness.

This study was conducted to investigate the diversity of phage types and associations between penicillin resistance and phage types among 815 Staphylococcus aureus isolates from bovine mastitis in nine European countries and USA. All isolates were examined for susceptibility to antimicrobial agents and characterised by phage typing. Penicillin resistance was found among strains from all countries with an average occurrence of 32.4% (2-71.4%). A total of 76% of isolates were identifiable by phage typing and 144 different phage types were observed. The most predominant types were phage type 29 (11% of the 815 isolates), phage type 52 (5%), and phage type 80 (5%). Phage type 95 and 29/52/52A/80 were both distributed within seven countries. In the countries with the highest occurrence of penicillin resistance a reduced diversity of phage types and phage groups was observed. Phage group In was significantly associated with penicillin resistance in contrast to phage group I (P = 0.0023) and phage complex-80 (P = 0.0066). This study confirms that a large number of phage types of S. aureus cause bovine mastitis, but that some types predominate. In addition, these findings could indicate that the use of penicillin in the bovine environment has selected for specific types of S. aureus in countries with a high frequency of resistance.

To the Editor: Salmonella enterica is among the leading causes of foodborne diseases worldwide. Multidrug-resistant S. enterica serovar Typhimurium definitive phage type (DT) 104 emerged during the early 1990s in the United Kingdom and spread worldwide thereafter (1). This phage-type strain harbors a chromosomally encoded genomic island, Salmonella Genomic Island 1, which is typically responsible for resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide, and tetracycline (2). Multilocus variable-number tandem-repeat analysis (MLVA) is an established molecular epidemiologic tool; its high-resolution power has been applied to the subtyping of a variety of bacterial species (3). An MLVA system has been developed for analyzing S. enterica serovar Typhimurium (4,5). The design of an MLVA system relies on the analyzed genome sequences. In this study, we found and evaluated a variable-number tandem-repeat region, or locus, designated DT104o. The locus is specific to S. enterica ser. Typhimurium DT104, according to the sequence of NCTC 13348 (available from the Sanger Institute, http://www.sanger.ac.uk/resources/downloads/bacteria/salmonella.html). The repeat unit sequence was CTCAGAA/TTCTGAG, spanning 1952121–1952274 on the reference genome or 22 repeats of 7 nt. We used 266 apparently independent isolates of S. enterica serovar Typhimurium collected during 1981–2012; 103 were from human samples and 163 from non-human sources. Bacteriophage typing was performed according to Anderson’s method and scheme (6). Types of 100 isolates were in the DT104 group, comprising DT104, DT104B, and U302, the latter being related to DT104 (2); MLVA was performed by using the 5 loci (STTR3, STTR5, STTR6, STTR9, and STTR10) with slight modifications (4,5). The DT104o locus was tested by using primers o-for (5′-GTCAACATGAACTGCCCCTCA-3′), labeled with NED, and o-rev (5′-TTTGCTCTTCGCTCTTAGCAATC-3′); this spanned 1952367–1952043 on the reference sequence, resulting in a 325-bp product with 171-bp offset. For all 266 isolates tested, the number of alleles and the Simpson’s index of diversity score (D) identified in each locus are summarized in the Table. The 5 common and DT104o loci displayed high discriminatory power: DT104o was specific for the DT104 group, and all 100 DT104 group isolates displayed amplified products with 13–40 repeat copy numbers; the others showed the null allele. Focusing only on the 100 DT104 group isolates, the discriminatory power of STTR9 and STTR3 were poor, whereas STTR5, STTR6, STTR10, and DT104o displayed high discriminatory powers (Table). In addition, using the 5 common loci (MLVA5) in analysis, we identified 66 types with a D value of 0.974; use of MLVA5 plus the DT104o locus (MLVA6) identified 83 types with a D value of 0.984. These results indicate that the DT104o locus is highly specific and therefore useful as an additional molecular epidemiologic marker for analyzing S. enterica ser. Typhimurium DT104. Table Number of alleles and Simpson's index of diversity score in Salmonella enterica serovar Typhimurium definitive phage type 104 in humans* Because DT104o was highly variable, 5 DT104 strains were tested for the frequency of variants at each locus after 5 serial passages by using liquid culture: cultures were diluted 1:1,000 at each passage. Sixteen colonies of each strain were tested by using MLVA6 (Technical Appendix Table 1). No variants were observed in STTR3, STTR9, or STTR10. STTR5, STTR6, and DT104o each showed 1 variant of 80 colonies. The results suggest that DT104o would not be less stable than other loci. We also found that DT104o could provide more discriminatory power to MLVA5 in some settings (Technical Appendix Table 2). We compared 2 settings using isolates from non-human samples. Setting 1 comprised isolates 1a and 1b from an outbreak during 1996 and isolate 1c in 2007. Isolates 1a and 1b were identical by MLVA6. Isolate 1c was identical by MLVA5 but not by MLVA6. In Setting 2, three isolates obtained in different years also were identical by MLVA5, but differed from each other by MLVA6. This suggests that MLVA6 could be useful in some epidemiologic settings such as in an outbreak investigation, though more extensive study would be required to confirm this suggestion. The DT104o locus is located at the proximal region of fragment 180 comprised of a prophage structure, which was proven to be DT104-specific in a previous study (7). This finding is consistent with the results of our study. In conclusion, development of an MLVA system is dependent upon the genome sequences available, and the system is usually used for molecular subtyping of a certain serotype in a particular organism. However, a specific group of strains could cause a pandemic and become a target of public health concern, as was S. enterica ser. Typhimurium DT104. The MLVA system could be improved by adding loci based on the genome sequence of such pandemic strains. In this study, we showed that the newly identified DT104o locus could be useful in identification and subtyping of S. enterica ser. Typhimurium DT104. Technical Appendix: Variation of locus and repeat copy number in settings identical in MLVA5 but not in MLVA6 in Salmonella enterica serovar Typhimurium definitive phage type 104 isolated from nonhuman samples. Click here to view.(57K, pdf)

Drug-resistant derivatives of serovar-specific virulence plasmids, such as pSLT, in clinically-relevant Salmonella enterica serovar Typhimurium strains, represent a threat for human health. We have analysed 14 S. Typhimurium isolates recovered in Italy and the United Kingdom from swine and from cases of human infection for the presence of virulence-resistance (VR) plasmids. They were negative for the multidrug resistance (MDR) region of the Salmonella genomic island 1 (SGI1), but expressed resistance to ampicillin, chloramphenicol, streptomycin/spectinomycin, sulfamethoxazole, and tetracyclines. The isolates were characterised by XbaI pulsed-field gel electrophoresis, multilocus sequence typing, and detection of resistance and virulence determinants (PCR/sequencing). Identification of VR plasmids was accomplished by PCR detection of bla genes (encoding ampicillin resistance), class 1 integrons and the pSLT virulence gene spvC. Plasmid analyses were performed by alkaline lysis, S1-nuclease digestion, replicon typing, conjugation, restriction analyses, and Southern blot/hybridization. Two blaOXA-1 positive isolates contained pSLT-derived plasmids related to pUO-StVR2. In nine isolates, eight from swine and one from a patient, MDR-conferring-IncFII-VR plasmids were detected. They contained the blaTEM-1 gene as well as a nonconventional class 1 integron with dfrA12-aadA2 gene cassettes in its variable region, and a sul3 gene in the 3' conserved segment. Restriction analysis suggested a novel pSLT variant. The results obtained underline the role of swine as a potential reservoir for the blaTEM-1-IncFII-plasmids. The occurrence and spread of virulence- and MDR-conferring plasmids should be considered as a potential public health problem.

Surveillance of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) is generally considered to benefit from molecular techniques like multiple-locus variable-number of tandem repeats analysis (MLVA), which allow earlier detection and confinement of outbreaks. Here, a surveillance study, including phage typing, antimicrobial susceptibility testing and the in Europe most commonly used 5-loci MLVA on 1,420 S. Typhimurium isolates collected between 2010 and 2012 in Belgium, was used to evaluate the added value of MLVA for public health surveillance. Phage types DT193, DT195, DT120, DT104, DT12 and U302 dominate the Belgian S. Typhimurium population. A combined resistance to ampicillin, streptomycin, sulphonamides and tetracycline (ASSuT) with or without additional resistances was observed for 42.5% of the isolates. 414 different MLVA profiles were detected, of which 14 frequent profiles included 44.4% of the S. Typhimurium population. During a serial passage experiment on selected isolates to investigate the in vitro stability of the 5 MLVA loci, variations over time were observed for loci STTR6, STTR10, STTR5 and STTR9. This study demonstrates that MLVA improves public health surveillance of S. Typhimurium. However, the 5-loci MLVA should be complemented with other subtyping methods for investigation of possible outbreaks with frequent MLVA profiles. Also, variability in these MLVA loci should be taken into account when investigating extended outbreaks and studying dynamics over longer periods.

Fourier transform infrared (FT-IR) spectroscopy and chemometric techniques were used to discriminate five closely related Salmonella enterica serotype Enteritidis phage types, phage type 1 (PT1), PT1b, PT4b, PT6, and PT6a. Intact cells and outer membrane protein (OMP) extracts from bacterial cell membranes were subjected to FT-IR analysis in transmittance mode. Spectra were collected over a wavenumber range from 4,000 to 600 cm −1 . Partial least-squares discriminant analysis (PLS-DA) was used to develop calibration models based on preprocessed FT-IR spectra. The analysis based on OMP extracts provided greater separation between the Salmonella Enteritidis PT1-PT1b, PT4b, and PT6-PT6a groups than the intact cell analysis. When these three phage type groups were considered, the method based on OMP extract FT-IR spectra was 100% accurate. Moreover, complementary local models that considered only the PT1-PT1b and PT6-PT6a groups were developed, and the level of discrimination increased. PT1 and PT1b isolates were differentiated successfully with the local model using the entire OMP extract spectrum (98.3% correct predictions), whereas the accuracy of discrimination between PT6 and PT6a isolates was 86.0%. Isolates belonging to different phage types (PT19, PT20, and PT21) were used with the model to test its robustness. For the first time it was demonstrated that FT-IR analysis of OMP extracts can be used for construction of robust models that allow fast and accurate discrimination of different Salmonella Enteritidis phage types.

Background Shiga toxin-producing Escherichia coli (STEC) have emerged as pathogens that can cause food-borne infections and severe and potentially fatal illnesses in humans, such as haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). In Spain, like in many other countries, STEC strains have been frequently isolated from ruminants, and represent a significant cause of sporadic cases of human infection. In view of the lack of data on STEC isolated from food in Spain, the objectives of this study were to determine the level of microbiological contamination and the prevalence of STEC O157:H7 and non-O157 in a large sampling of minced beef collected from 30 local stores in Lugo city between 1995 and 2003. Also to establish if those STEC isolated from food possessed the same virulence profiles as STEC strains causing human infections. Results STEC were detected in 95 (12%) of the 785 minced beef samples tested. STEC O157:H7 was isolated from eight (1.0%) samples and non-O157 STEC from 90 (11%) samples. Ninety-six STEC isolates were further characterized by PCR and serotyping. PCR showed that 28 (29%) isolates carried stx1 genes, 49 (51%) possessed stx2 genes, and 19 (20%) both stx1 and stx2. Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 43 (45%) and in 25 (26%) of the isolates, respectively. Typing of the eae variants detected four types: γ1 (nine isolates), β1 (eight isolates), ε1 (three isolates), and θ (two isolates). The majority (68%) of STEC isolates belonged to serotypes previously detected in human STEC and 38% to serotypes associated with STEC isolated from patients with HUS. Ten new serotypes not previously described in raw beef products were also detected. The highly virulent seropathotypes O26:H11 stx1eae-β1, O157:H7 stx1stx2eae-γ1 and O157:H7 stx2eae-γ1, which are the most frequently observed among STEC causing human infections in Spain, were detected in 10 of the 96 STEC isolates. Furthermore, phage typing of STEC O157:H7 isolates showed that the majority (seven of eight isolates) belonged to the main phage types previously detected in STEC O157:H7 strains associated with severe human illnesses. Conclusion The results of this study do not differ greatly from those reported in other countries with regard to prevalence of O157 and non-O157 STEC in minced beef. As we suspected, serotypes different from O157:H7 also play an important role in food contamination in Spain, including the highly virulent seropathotype O26:H11 stx1eae-β1. Thus, our data confirm minced beef in the city of Lugo as vehicles of highly pathogenic STEC. This requires that control measures to be introduced and implemented to increase the safety of minced beef.

Objective To examine 13 Salmonella typhimurium and 22 S. enteritidis strains isolated from individual cases of gastroenteritis for their phage types, antibiotic susceptibilities and plasmid profiles. Methods The phage typing of S. typhimurium strains was done according to the method of Anderson et al, and the phage typing scheme of Ward et al was used for phage typing of S. enteritidis strains. Antibiotic susceptibility testing was performed by the Kirby–Bauer disk diffusion method. Extended-spectrum β-lactamase production of the strains was determined by the three-dimensional method. Plasmid profiles of the strains were examined using the method described by Kado and Liu with some modification by Graeber et al. Results Two S. typhimurium strains were DT 193 and one was DT 22, whereas 10 strains were untypable. PT 4 was the predominant phage type among S. enteritidis strains. Four S. enteritidis strains were DT 6a, three strains were PT 1 and one strain was PT 8, whereas only one strain was untypable. Eleven of 13 S. typhimurium and three of 22 S. enteritidis strains were found to be multiresistant. Ten different resistance patterns among S. typhimurium and four different resistance patterns among S. enteritidis strains were detected. Extended-spectrum β-lactamase production was detected in 10 of 13 S. typhimurium and in three of 22 S. enteritidis strains. All S. typhimurium strains but one were found to contain at least one plasmid, with molecular masses varying between 4 and 107 MDa, and 11 different plasmid patterns were determined. Plasmid pattern analysis permitted further differentiation of the S. enteritidis strains into nine groups. A serovar-specific virulence plasmid of 36 MDa was detected in 13 of 22 S. enteritidis strains. Conclusions The results suggest that the majority of S. typhimurium strains were closely related.

Background Salmonella enterica serovar Typhimurium (or simply Typhimurium) is the most common serovar in both human infections and farm animals in Australia and many other countries. Typhimurium is a broad host range serovar but has also evolved into host-adapted variants (i.e. isolated from a particular host such as pigeons). Six Typhimurium strains of different phage types (defined by patterns of susceptibility to lysis by a set of bacteriophages) were analysed using Illumina high-throughput genome sequencing. Results Variations between strains were mainly due to single nucleotide polymorphisms (SNPs) with an average of 611 SNPs per strain, ranging from 391 SNPs to 922 SNPs. There were seven insertions/deletions (indels) involving whole or partial gene deletions, four inactivation events due to IS200 insertion and 15 pseudogenes due to early termination. Four of these inactivated or deleted genes may be virulence related. Nine prophage or prophage remnants were identified in the six strains. Gifsy-1, Gifsy-2 and the sopE2 and sspH2 phage remnants were present in all six genomes while Fels-1, Fels-2, ST64B, ST104 and CP4-57 were variably present. Four strains carried the 90-kb plasmid pSLT which contains several known virulence genes. However, two strains were found to lack the plasmid. In addition, one strain had a novel plasmid similar to Typhi strain CT18 plasmid pHCM2. Conclusion The genome data suggest that variations between strains were mainly due to accumulation of SNPs, some of which resulted in gene inactivation. Unique genetic elements that were common between host-adapted phage types were not found. This study advanced our understanding on the evolution and adaptation of Typhimurium at genomic level.