Although milk and milk products have been represented as an important vehicle
for the foodborne disease transmission to humans, in developing countries; limited
publications were documented shigellosis outbreaks in related to the consumption of
milk and milk products. The current study showed a high prevalence of Shigella spp. (7%) with the predominance of S. dysenteriae in comparison to those recorded by Ahmed and Shimamoto who reported thatShigella spp. were detected in 0.5% of raw cows milk samples and 0.9% of Kareish cheese samples
with S. felxeneri as the most predominant isolates.
Bhutdadetected 8.7% of S. flexneri in milk product (pedha) samples in India. The high rate of Shigella prevalence in this study might be regarded to poor hygienic measures during milking,
processing, preparation, handling, and storage of milk and milk products.
Antibiotic resistance of Shigella spp. from raw milk and milk products samples in this study was compared with previous
reports from Egypt to observe the trend in antibiotic resistance keeping in mind that
the respective study samples collection. Overall, this study had displayed the entity
of warning levels of Shigella spp. resistant to the prevalently used antibiotics (tetracycline, ampicillin, amoxicillin-clavulanate,
cefaclor) [21,22]. This resistance might be due to the fact of frequently and improperly using such
antibiotics either in animal therapy or as a growth promoter in the veterinary context
in Egyp. Also, the current study showed reduced susceptibility to cefotaxime, ceftazidime
(third-generation cephalosporins) and ciprofloxacin, which were considered as preferable
drugs for shigellosis treatment . Thus, the appearance of such resistance would indicate a great challenge for the
efficient treatment of shigellosis.
In this study, about 71.4% of Shigella isolates were multidrug-resistant to at least three of the antimicrobial classes
with multidrug antibiotic resistance index (MARI) of 0.2-0.5. In addition, many isolates
of Shigella spp. were shown to have MR phenotypes against tetracycline, ampicillin, amoxicillin-clavulanate,
and cefaclor pattern. Nearly similarity, the high prevalence of MR Shigella isolates in dairy products (90.9%) was reported by Ahmed and Shimamoto in Egypt. Therefore, some measures must be considered to confirm that currently available
antibiotics remain effective. These measures may include great awareness among the
public, healthcare professionals and the food-agriculture sector concerning the importance
of the proper use of these medicines.
The presence of Shigella in the analyzed samples was an indicator of poor hygiene and sanitation during milking,
post-milking and during milk processing. The effectiveness of the disinfection depended
on the use of suitable disinfectant which was considered from the most critical points
of hygienic measures in dairy cattle farms. The phenolic compounds and benzalkonium chloride (BKC) were widely used as farm disinfectant due to their antimicrobial activity . BKC is a cationic, surface-active QAC commonly used as a farm disinfectant for cleaning and sanitizing of livestock buildings, equipment, milk utensils, and
vehicle. The present study exhibited that all the tested strains were resistant to phenolic
compound, while 85.7% of isolates were BKC resistance. Similarly, Bouzada et al. found that Gram-negative rods involving Enterobacteriaceae were less susceptible to
BKC. Moreover, this work displayed that most of the Shigella isolates (85.7%) had qacE∆1 gene.
There are various β-lactamases, which hydrolyses the β-lactam ring and thereby inactivate
the antibiotic, have been described, but TEM-, OXA-, SHV-and CTX-M-type β-lactamases
are dominant in the Gram-negative bacteria . Thus, in this investigation, the presence of these β-lactamase-encoding genes in
isolates was recognized by molecular methods, which provided data to support the current
study. The blaTEM, a narrow-spectrum β-lactamase gene, which conveys resistance to penicillins and first-generation cephalosporins,
was identified in all isolates. Also, the extended spectrum β-lactamase-encoding genes blaCTX-Mwas identified in 28.6% of the isolates. The high incidence of β-lactamase-encoding
genes (blaTEM-1, blaCTX-M, in 2 isolates, blaOXA, in 4 isolates) had been detected previously in Shigella strains isolated from dairy products in Egypt . The blaTEMgene was the dominant β-lactamase gene in Shigella spp. in this work, while blaCTX-Mwas the most common types of cefotaximases identified among Shigella isolates in the previous study .
Alarmingly, in this research, the prevalence of ESBL producing Shigella isolates, accounting for 28.6% of all Shigella isolates, was higher than detecting rates observed in other countries such as England
. This discrepancy between such finding and previous studies might be attributed to
the misuse of antibiotics during the treatment of bacterial infections. In addition,
the high tetracycline resistance in all Shigella isolates might be described by the potential distribution of the tetA(A) resistance gene .
For epidemiological investigations of various enteric pathogens, plasmid profiling
could be an attractive tool. Shigella usually harbor various plasmids that might be from 2 to 10 plasmids in one strain.
These plasmids were required for antibiotic resistance and also for invading of bacteria
into the epithelial cells of the intestine . Seven Plasmid Patterns, with relative plasmid sizes ranging from 1.26 to 33.61 kb,
were detected in this study. It had been reported earlier that Shigella species had numbers of plasmids with a heterogeneous combination ranging from 1.0
to 120 MDa in Egypt . Also, some of the plasmid sizes documented among the Shigella isolates were previously reported in Egypt and other countries [30,31,32]. All MR strains particularly ESBL producing strains carried plasmids with pattern
P7 as the predominant pattern in this study. The ESBL encoding gene (blaCTX-M) had been conveyed to plasmid more than other Class A β-lactamases . The conjugation experiment results assisted the plasmid location of blaTEM, blaCTX-M, and qacE∆1 genes, since transconjugants of MR phenotypes of Shigella isolates were done on MacConkey agar. Of the 12 S. dysenteriae isolates in this work, 75% contained large plasmids, which were associated with antibiotic
resistance. Over the last half-century, the extraordinary ability of different isolates
in acquiring plasmid-encoded resistance to the disinfectants and antibiotics such
as ESBLs, that could quickly be transmitted to several other strains,had demonstrated [34,35].
Antibiotics and disinfectants had been commonly used in dairy farms in Egypt. However,
the susceptibility of Shigella to disinfectant and its contribution to multidrug resistance phenotype and genotype
by co-selection of plasmid had never been reported. Plasmid-mediated multidrug resistance
should be considered for dealing with infectious diseases. Therefore, this work was
planned to explore the link between qacE∆1 gene, plasmid, and antibiotic resistance. The results of this study showed that all qacE∆1gene positive strains were multi-resistant and plasmid harbored. Recently, it was
demonstrated that the qacC gene conveys resistance to a number of β-lactam antibiotics . While it was found the ability of qac genes to directly acquire resistance to antibiotics. This indicated the close relationship
between the resistance to antibiotics and antiseptics . An approaching interdependence between resistance to antiseptics and antibiotics
might be principally elucidated by the fact that genetic determinants of resistance
to these agents were mainly associated. The plasmids frequently conveyed qac genes with a number of other antibiotic resistance genes [38,39]. The plasmid analysis and conjugation experiments showed that the isolates harbored
variable detectable plasmids and the antibiotic and disinfectant resistance genes
could be co-transferred. This indicated that the resistance was plasmid-mediated,
so there was a high risk for the spread of antibiotic and disinfectant resistance
genes among the bacteria.